U.S. patent number 8,343,501 [Application Number 12/625,217] was granted by the patent office on 2013-01-01 for bispecific egfr/igfir binding molecules.
This patent grant is currently assigned to Bristol-Myers Squibb Company. Invention is credited to Ray Camphausen, Joan Carboni, Ginger Chao, Stuart Emanuel, Linda Engle, Marco Gottardis, Martin C. Wright.
United States Patent |
8,343,501 |
Emanuel , et al. |
January 1, 2013 |
**Please see images for:
( Certificate of Correction ) ** |
Bispecific EGFR/IGFIR binding molecules
Abstract
The present invention relates to bispecific molecules comprising
an EGFR binding domain and a distinct IGFIR binding domain for use
in diagnostic, research and therapeutic applications. The invention
further relates to cells comprising such proteins, polynucleotide
encoding such proteins or fragments thereof, and vectors comprising
the polynucleotides encoding the innovative proteins. Exemplary
bispecific molecules include antibody-like protein dimers based on
the tenth fibronectin type III domain.
Inventors: |
Emanuel; Stuart (Doylestown,
PA), Engle; Linda (Framingham, MA), Camphausen; Ray
(Wayland, MA), Wright; Martin C. (Boston, MA), Chao;
Ginger (Cambridge, MA), Gottardis; Marco (Princeton,
NJ), Carboni; Joan (Yardley, PA) |
Assignee: |
Bristol-Myers Squibb Company
(Princeton, NJ)
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Family
ID: |
41667546 |
Appl.
No.: |
12/625,217 |
Filed: |
November 24, 2009 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20100179094 A1 |
Jul 15, 2010 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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61200164 |
Nov 24, 2008 |
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61200282 |
Nov 26, 2008 |
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61212966 |
Apr 17, 2009 |
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61178279 |
May 14, 2009 |
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61227330 |
Jul 21, 2009 |
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Current U.S.
Class: |
424/185.1;
424/193.1; 514/19.2; 530/402; 514/9.3; 530/350 |
Current CPC
Class: |
A61P
35/00 (20180101); C07K 14/475 (20130101); A61K
45/06 (20130101); A61P 43/00 (20180101); C07K
14/78 (20130101); A61K 38/39 (20130101); C07K
16/2863 (20130101); A61K 39/3955 (20130101); A61K
2039/505 (20130101); C07K 2317/92 (20130101); C07K
2319/30 (20130101); C07K 2317/34 (20130101); C07K
2318/20 (20130101); C07K 2317/31 (20130101) |
Current International
Class: |
A61K
38/17 (20060101); C07K 14/71 (20060101); C07K
14/435 (20060101); A61K 47/48 (20060101); A61K
38/39 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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WO 00/34784 |
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Jun 2000 |
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WO |
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WO 01/64942 |
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Sep 2001 |
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WO |
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WO 02/32925 |
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Apr 2002 |
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WO |
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WO 2005/056764 |
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Jun 2005 |
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WO |
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WO 2006/020258 |
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Feb 2006 |
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WO |
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WO 2007/012614 |
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Feb 2007 |
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WO |
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WO 2008/066752 |
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Jun 2008 |
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WO |
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WO 2008/097497 |
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Aug 2008 |
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WO |
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WO 2008/108986 |
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Sep 2008 |
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WO |
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WO 2009/025806 |
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Feb 2009 |
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WO |
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WO 2009/073115 |
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Jun 2009 |
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WO |
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WO 2009/102421 |
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Aug 2009 |
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WO |
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WO 2009/142773 |
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Nov 2009 |
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WO |
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Primary Examiner: Bunner; Bridget E
Attorney, Agent or Firm: Nelson Mullins Riley &
Scarborough LLP Remillard, Esq.; Jane E. Kanik; Cynthia L.
Parent Case Text
RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Application
Nos. 61/200,164, filed Nov. 24, 2008; 61/200,282, filed Nov. 26,
2008; 61/212,966, filed Apr. 17, 2009; 61/178,279, filed May 14,
2009; and 61/227,330, filed Jul. 21, 2009, which applications are
hereby incorporated by reference in their entireties.
Claims
We claim:
1. An antibody-like protein dimer comprising (a) a tenth
fibronectin type III domain (.sup.10Fn3) that binds insulin-like
growth factor 1 receptor (IGF-IR) with a K.sub.D of less than 500
nM covalently or non-covalently linked to (b) a .sup.10Fn3 that
binds epidermal growth factor receptor (EGFR) with a K.sub.D of
less than 500 nM; wherein the IGF-IR binding .sup.10Fn3 comprises a
BC loop having the amino acid sequence set forth in amino acids
21-30 of SEQ ID NO: 3, a DE loop having the amino acid sequence set
forth in amino acids 51-56 of SEQ ID NO: 3, and an FG loop having
the amino acid sequence set forth in amino acids 76-83 of SEQ ID
NO: 3, and wherein the EGFR binding .sup.10Fn3 comprises a BC loop
having the amino acid sequence set forth in amino acids 21-30 of
SEQ ID NO: 112, a DE loop having the amino acid sequence set forth
in amino acids 51-56 of SEQ ID NO: 112, and an FG loop having the
amino acid sequence set forth in amino acids 76-92 of SEQ ID NO:
112.
2. The antibody-like protein dimer of claim 1, wherein the IGF-IR
binding .sup.10Fn3 is covalently linked to the EGFR binding
.sup.10Fn3 via a polypeptide linker or a polyethylene glycol
moiety.
3. The antibody-like protein dimer of claim 2 comprising an amino
acid sequence at least 90% identical to any one of SEQ ID NOs:
126-132.
4. The antibody-like protein dimer of claim 3, comprising an amino
acid sequence at least 90% identical to SEQ ID NO: 130.
5. The antibody-like protein dimer of claim 4, comprising an amino
acid sequence at least 95% identical to SEQ ID NO: 130.
6. The antibody-like protein dimer of claim 5, comprising an amino
acid sequence at least 98% identical to SEQ ID NO: 130.
7. The antibody-like protein dimer of claim 6, further comprising a
C-terminal tail comprising the amino acid sequence of SEQ ID NO:
217.
8. The antibody-like protein dimer of claim 7, further comprising
one or more pharmacokinetic (PK) moieties.
9. The antibody-like protein dimer of claim 8, wherein the PK
moiety is a polyoxyalkylene moiety and said polyoxyalkylene moiety
is polyethylene glycol.
10. The antibody-like protein dimer of claim 9, wherein the
polyethylene glycol is between 0.1 kDa and 150 kDa.
11. The antibody-like protein dimer of claim 10, wherein the
polyethylene glycol is linked to the antibody-like protein dimer
via a Cys amino acid residue.
12. The antibody-like protein dimer of claim 6, comprising the
amino acid sequence set forth in SEQ ID NO: 130.
13. An antibody-like protein dimer comprising a tenth fibronectin
type III domain (.sup.10Fn3 ) that binds insulin-like growth factor
1 receptor (IGF-IR) with a K.sub.D of less than 500 nM covalently
or non-covalently linked to a .sup.10Fn3 that binds epidermal
growth factor receptor (EGFR) with a K.sub.D of less than 500 nM;
wherein the IGF-IR binding .sup.10Fn3 comprises a BC loop having
the amino acid sequence X.sub.aSARLKVAX.sub.b (SEQ ID NO: 46), a DE
loop having the amino acid sequence X.sub.CKNVYX.sub.d (SEQ ID NO:
48), and an FG loop having the amino acid sequence
X.sub.eRFRDYQX.sub.f (SEQ ID NO: 50); wherein the EGFR binding
.sup.10Fn3 comprises a BC loop having the amino acid sequence
X.sub.gWAPVDRYQX.sub.h (SEQ ID NO: 137), a DE loop having the amino
acid sequence X.sub.iRDVYX.sub.j (SEQ ID NO: 138), and an FG loop
having the amino acid sequence X.sub.kDYKPHADGPHTYHESX.sub.l (SEQ
ID NO: 139); and wherein X is any amino acid and a, b, c, d, e, f,
g, h, i, j, k, and l are integers independently selected from 0 to
5.
14. The antibody-like protein dimer of claim 13, wherein the IGF-IR
binding .sup.10Fn3 comprises a BC loop having the amino acid
sequence SWSARLKVAR (SEQ ID NO: 45), a DE loop having the amino
acid sequence PKNVYT (SEQ ID NO: 47), and an FG loop having the
amino acid sequence TRFRDYQP (SEQ ID NO: 49); and an EGFR binding
.sup.10Fn3 comprising a BC loop having the amino acid sequence
SWWAPVDRYQ (SEQ ID NO: 115), a DE loop having the amino acid
sequence PRDVYT (SEQ ID NO: 116), and an FG loop having the amino
acid sequence TDYKPHADGPHTYHESP (SEQ ID NO: 117).
15. The antibody-like protein dimer of any one of claims 1-14,
wherein the IGF-IR binding .sup.10Fn3 has an amino acid sequence at
least 90% identical to SEQ ID NO: 3 and the EGFR binding .sup.10Fn3
has an amino acid sequence at least 90% identical to SEQ ID NO:
112.
16. The antibody-like protein dimer of claim 15, wherein the IGF-IR
binding .sup.10Fn3 has an amino acid sequence at least 95%
identical to SEQ ID NO: 3 and the EGFR binding .sup.10Fn3 has an
amino acid sequence at least 95% identical to SEQ ID NO: 112.
17. The antibody-like protein dimer of claim 16, wherein the IGF-IR
binding .sup.10Fn3 has an amino acid sequence at least 98%
identical to SEQ ID NO: 3 and the EGFR binding .sup.10Fn3 has an
amino acid sequence at least 98% identical to SEQ ID NO: 112.
18. The antibody-like protein dimer of claim 17, wherein the IGF-IR
binding .sup.10Fn3 has the amino acid sequence of SEQ ID NO: 3 and
the EGFR binding .sup.10Fn3 has the amino acid sequence of SEQ ID
NO: 112.
19. A pharmaceutically acceptable composition comprising the
antibody-like protein dimer of any one of claim 1-14, 2-3, or 4-10,
wherein the composition is essentially endotoxin free.
Description
SEQUENCE LISTING
The instant application contains a Sequence Listing which has been
submitted via EFS-Web and is hereby incorporated by reference in
its entirety. Said ASCII copy, created on Nov. 23, 2009, is named
COTH5261.txt, and is 511,653 bytes in size.
FIELD OF THE INVENTION
The present invention relates to EGFR binding domains and
bispecific molecules comprising an EGFR binding domain and a
distinct IGFIR binding domain for use in diagnostic, research and
therapeutic applications. The invention further relates to cells
comprising such proteins, polynucleotide encoding such proteins or
fragments thereof, and vectors comprising the polynucleotides
encoding the innovative proteins. Exemplary EGFR binding domains
and bispecific molecules include antibody-like protein dimers based
on the tenth fibronectin type III domain.
INTRODUCTION
Activation of receptor tyrosine kinase signaling is central to
cancer development (see e.g., Grimberg A. Cancer Biol Ther. 2003
2(6):630-5 and Mendelsohn J. J Clin Oncol. 2003 21(14):2787-99).
Receptor tyrosine kinases have a conserved domain structure
including an extracellular domain, a transmembrane domain and an
intracellular tyrosine kinase domain. The extracellular domain can
bind to a ligand, such as to a polypeptide growth factor or to a
cell membrane-associated molecule. Typically, either ligand binding
or ligand binding induced dimerization of receptor tyrosine kinases
activates the intracellular catalytic tyrosine kinase domain of the
receptor and subsequent signal transduction.
Examples of receptor tyrosine kinases include, but are not limited
to ERBB receptors (e.g., EGFR, ERBB2, ERBB3, ERBB4),
erythropoietin-producing hepatocellular (EPH) receptors, fibroblast
growth factor (FGF) receptors (e.g., FGFR1, FGFR2, FGFR3, FGFR4,
FGFR5), platelet-derived growth factor (PDGF) receptors (e.g.,
PDGFR-A, PDGFR-B), vascular endothelial growth factor (VEGF)
receptors (e.g., VEGFR1/FLT1, VEGFR2/FLK1, VEGF3), tyrosine kinase
with immunoglobulin-like and EGF-like domains (TIE) receptors,
insulin-like growth factor (IGF) receptors (e.g., INS-R, IGFIR,
IR-R), Discoidin Domain (DD) receptors, receptor for c-Met (MET),
recepteur d'origine nantais (RON); also known as macrophage
stimulating 1 receptor, Flt3 fins-related tyrosine kinase 3 (Flt3),
colony stimulating factor 1 (CSF1) receptor, adhesion related
kinase receptor (e.g., Axl), receptor for c-kit (KIT) and insulin
receptor related (IRR) receptors.
Inhibition of receptor tyrosine kinases has emerged as an effective
treatment strategy for certain human malignancies (for a review see
Roussidis A E, In Vivo. 2002 16(6):459-69). While targeted
monotherapy may initially be effective in treating cancer,
therapeutic resistance often follows, possibly as a result of
upregulation of other signaling cascades (see e.g., Nahta R et al.,
Breast Cancer Res. 2006 8(6):215 and Horn L et al., Clin Lung
Cancer. 2007 8:S68-73). Accordingly, there exists a need for
developing improved cancer therapeutics.
SUMMARY OF THE INVENTION
In one aspect, the application provides EGFR binding tenth
fibronectin type III domains (.sup.10Fn3) having novel sequences.
EGFR binding .sup.10Fn3 having a consensus sequence are also
provided. Such EGFR binding .sup.10Fn3 may be monomeric or may be
included as part of a fusion protein.
In another aspect, the application provides bispecific molecules
that bind EGFR and IGFIR, referred to herein as "E/I binders". E/I
binders encompassed by the invention include bispecific antibodies
and dimers of ligand binding scaffold proteins (e.g., tendamistat,
affibody, fibronectin type III domain, anticalin, tetranectin, and
ankyrin). When constructed as a single polypeptide chain, the E/I
binders may be constructed in any orientation, e.g., from
N-terminus to C-terminus either in the E-I arrangement or the I-E
arrangement.
In one aspect, antibody-like protein dimers are provided comprising
an EGFR binding .sup.10Fn3 covalently or non-covalently linked to
an IGFIR binding .sup.10Fn3. The .sup.10Fn3 bind their target (EGFR
or IGFIR) with a K.sub.D of less than 500 nM. Each of the
individual .sup.10Fn3 independently has an amino acid sequence at
least 70, 80, 85, 90, 95, 98, or 100% identical to SEQ ID NO: 32,
wherein n is an integer from 1-20, o is an integer from 1-20, and p
is an integer from 1-40. In some embodiments, n is an integer from
8-12, o is an integer from 4-8, and p is an integer from 4-28. In
some embodiments, n is 10, o is 6, and p is 12.
In some embodiments, the antibody-like protein dimers comprise
IGFIR binding .sup.10Fn3 covalently linked to EGFR binding
.sup.10Fn3 via a polypeptide linker or a polyethylene glycol
moiety. In some embodiments, the antibody-like protein dimer
comprises an amino acid sequence at least 80, 90, 95, or 100%
identical to any one of SEQ ID NOs: 20-31, 53-58, 87-92, 98-105,
118-133, 149-154, 164-169, 179-184, 192-197, 205-210 and
211-216.
In some embodiments, the E/I binder comprises an amino acid
sequence having any one of SEQ ID NOs: 20-31, 53-58, 87-92, 98-105,
118-133, 149-154, 164-169, 179-184, 192-197, 205-210 and 211-216,
wherein (i) the EGFR binding .sup.10Fn3 and/or the IGF-IR binding
.sup.10Fn3 comprises a .sup.10Fn3 scaffold having from has anywhere
from 0 to 20, from 0 to 15, from 0 to 10, from 0 to 8, from 0 to 6,
from 0 to 5, from 0 to 4, from 0 to 3, from 0 to 2, or from 0 to 1
substitutions, conservative substitutions, deletions or additions
relative to the corresponding scaffold amino acids of SEQ ID NO: 1,
and/or (ii) the EGFR binding .sup.10Fn3 has anywhere from 0 to 15,
from 0 to 10, from 0 to 8, from 0 to 6, from 0 to 5, from 0 to 4,
from 0 to 3, from 0 to 2, or from 0 to 1 substitutions,
conservative substitutions, deletions or additions relative to the
corresponding loop sequences of any one of SEQ ID NOs: 5-8, 52,
66-68, 106-108, 112-114, 140-142, 155-157, 170-172, 182, 185-187,
198-200, or 219-327 and/or the IGF-IR binding .sup.10Fn3 has
anywhere from 0 to 15, from 0 to 10, from 0 to 8, from 0 to 6, from
0 to 5, from 0 to 4, from 0 to 3, from 0 to 2, or from 0 to 1
substitutions, conservative substitutions, deletions or additions
relative to the corresponding loop sequences of SEQ ID NO: 3.
In one aspect, pharmaceutically acceptable compositions are
provided comprising an antibody-like protein dimer as described
herein and a pharmaceutically acceptable carrier, wherein the
composition is essentially pyrogen free.
In a further aspect, methods for treating hyperproliferative
disorders, such as cancer, in a subject are provided comprising
administering to a subject in need thereof a therapeutically
effective amount of a pharmaceutically acceptable composition
comprising an antibody-like protein dimer as described herein.
In another aspect, the application provides a nucleic acid encoding
an antibody-like protein dimer as described herein. Also provided
is a vector comprising a nucleic acid encoding an antibody-like
dimer as described herein. Suitable vectors include, for example,
expression vectors. Also provided are host cells comprising a
nucleic, vector, or expression vector, comprising a nucleic acid
encoding an antibody-like protein dimer as described herein.
Suitable host cells include prokaryotic and eukaryotic host cells.
Exemplary prokaryotic cells are bacterial cells, such as E. coli.
Exemplary eukaryotic cells are mammalian cells, such as CHO cells.
Also provided are methods for producing an antibody-like protein
dimer as described herein, comprising culturing a host cell
comprising a nucleic, vector, or expression vector, comprising a
nucleic acid encoding the antibody-like protein dimer and
recovering the expressed antibody-like protein dimer from the
culture.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. SDS-PAGE Analysis of I1-GS10-E2. Samples from the lysis of
HMS174(DE3) bacterial cell pellet from which I1-GS10-E2 was
expressed and purified by a HisTrap chromatography column were run
on a 4-12% NuPAGE minigel and stained by Sypro-Orange and
visualized by STORM imager. Mark 12 molecular weight standards
(Lane 1); Lysate-soluble (Lane 2); Lysate-insoluble (Lane 3);
HisTrap load (Lane 4); HisTrap non-bound (Lane 5); Pooled HisTrap
Eluate (Lane 6); Dialyzed into 50 mM NaOAc, 150 mM NaCl, pH 4.5
(Lane 7); Dialyzed into PBS (Lane 8); Dialyzed into Tris, 150 mM
NaCl, pH 8.5 (Lane 9).
FIG. 2. A. SEC Analysis of midscale purified I1-GS10-E2. 22 .mu.g
of HisTrap purified I1-GS10-E2 dialyzed into PBS, pH 7.4 was loaded
onto a Superdex 200 10/30 SEC Column (GE Healthcare) with a mobile
phase of 100 mM NaPO.sub.4, 100 mM NaSO.sub.4, 150 mM NaCl, pH 6.8
and measured using A280. I1-GS10-E2 eluted predominantly as a
single monomeric species at a molecular weight range of
approximately 24.6 kDa vs. globular Gel Filtration standards
(BioRad). B. SEC analysis of E2-GS10-I1.
FIG. 3. A. Differential Scanning Calorimetry (DSC) of midscale
purified I1-GS10-E2 in PBS was performed to determine the T.sub.m.
A 1 mg/mL solution of I1-GS10-E2 was scanned from 5.degree. C. to
95.degree. C. at a rate of 1 degree per minute under 3 atm
pressure. The data was analyzed versus a control run of the PBS
buffer. B. DSC of E2-GS10-I1.
FIG. 4. Inhibition of IGFR activity in H292 cells. Cells were
stimulated with 100 ng/mL of IGF-1 and 100 ng/mL of EGF and treated
with either .circle-solid. I1, .quadrature. E1, or .DELTA.
E1-GS10-HTPP preparations. Phosphorylation of IGFIR on tyrosine
1131 was determined by ELISA.
FIG. 5. Inhibition of EGFR activity in H292 cells. Cells were
stimulated with 100 ng/mL of IGF-1 and 100 ng/mL of EGF and treated
with either .circle-solid. I1, .quadrature. E1, or .DELTA.
E1-GS10-I1 HTPP preparations. Phosphorylation of EGFR on tyrosine
1068 was determined by ELISA.
FIG. 6. Inhibition of AKT phosphorylation in H292 cells. Cells were
stimulated with 100 ng/mL of IGF-1 and 100 ng/mL of EGF and treated
with either .circle-solid. I1, .quadrature. E1, or .DELTA.
E1-GS10-I1 HTPP preparations. Phosphorylation of AKT on serine 473
was determined by ELISA.
FIG. 7. Inhibition of RH41 cell proliferation. Cells were treated
with either .circle-solid. I1, .quadrature. E1, or .DELTA.
E1-GS10-I1 HTPP preparations and percent inhibition of
proliferation was determined.
FIG. 8. Inhibition of H292 cell proliferation. Cells were treated
with either .circle-solid. I1, .quadrature. E1, or .DELTA.
E1-GS10-I1 HTPP preparations and percent inhibition of
proliferation was determined.
FIG. 9. Summarizes IC50 values in cell based functional assays for
isolated EGFR mononectins, E/I .sup.10Fn3-based binders with serine
at the C-terminal position without PEG added and E/I
.sup.10Fn3-based binders with cysteine at the C-terminal position
conjugated to a 40 kDa branched PEG. Representative data is
shown.
FIG. 10. Immunoblot analysis of PEGylated E/I .sup.10Fn3-based
binder with E2 in the N-terminal and C-terminal positions. Despite
both constructs demonstrating comparable activity in the H292 cell
assay for inhibiting EGFR, the E/I .sup.10Fn3-based binder with E2
at the C-terminal position did not degrade EGFR while the E/I
.sup.10Fn3-based binder with E2 at the N-terminal position did.
Both constructs show very weak to no IGFR degradation in this cell
line. .beta.-actin was included to demonstrate equal loading across
all lanes. The phosphorylation state of EGFR, ERK and Shc was also
examined.
FIG. 11. Inhibition of EGF-stimulated EGFR phosphorylation in H292
cells. Both constructs demonstrated comparable activity in the H292
cell assay for inhibiting EGFR. E2-GS10-I1 (with PEG)
(.largecircle.), I1-GS10-E2 (with PEG) (.quadrature.), panitumumab
(----).
FIG. 12. Results of tumor xenograft studies. FIG. 12A: Preclinical
anti-tumor activity in the H292 human tumor xenograft model. Mean
tumor sizes calculated from groups of 8 mice is shown in mg for
control animals (.box-solid.), E3-GS10-I1 (w/PEG) dosed at 100
mg/kg (.largecircle.), E2-GS10-I1 (with PEG) dosed at 100 mg/kg
(.quadrature.), panitumumab dosed at 1 mg/mouse (.DELTA.) or 0.1
mg/mouse (.gradient.). The letter a on the x-axis indicates doses
of E/I binders administered and the p indicates doses of
panitumumab administered. FIG. 12B: Average weight change is shown
for each group over the course of the study. Symbols are as
described in FIG. 12A legend.
FIG. 13. Pharmacodynamic effects in the H292 NSCLC tumor xenograft
model. Levels of the indicated analytes were determined in tumor
lysates as described in Example 12. (A) phosph-EGFR, (B)
phospho-ErbB2, (C) phospho-IGFR, and (D) total EGFR. Checkered
bars=panitumumab, empty bars=E2-GS10-I1 (with PEG), hatched
bats=E3-GS10-I1 (with PEG).
FIG. 14. Western blot analysis of MCF7r cells compared to MCF7
parental cells.
FIG. 15. MCF7 (Panel A) and MCF7r (Panel B) human tumor xenograft
studies in nude mice. Mean tumor size is shown for both studies
calculated from 8 mice per group.
FIG. 16. GEO human tumor xenograft studies in nude mice.
FIG. 17. H292 human tumor xenograft studies in nude mice.
FIG. 18. Colony formation assay with H292 NSCLC cells. A.
Representative data is shown from a single plate. B. IC50 from one
E/I .sup.10Fn3-based binder is shown with error bars calculated
from triplicate measurements.
FIG. 19. Epitope mapping assay. Location of epitope binding for
various EGFR binding antibodies are shown in panel A. A description
of the antibodies is provided in Example 18, Table 11. The left
column of table 11 provides a number for each anti-EGFR antibody
which correlates with the numbered antibodies shown in panel A.
Panel B shows an exemplary epitope mapping assay as described in
Example 18.
FIG. 20. DSC analysis of the E/I .sup.10Fn3-based binder,
I1-GS10-E5 pegylated, measured with a scan range of 15-95.degree.
C. at 1 mg/ml protein concentration in PBS, resulted in a Tm
measurement of 55.2.degree. C.
FIG. 21. Evaluation of E/I .sup.10Fn3-based binders for inhibition
of AKT phosphorylation in H292 cells as measured by ELISA.
I1-GS10-E5-pegylated (.largecircle.) was more potent than
I1-pegylated alone (.box-solid.) or E5-pegylated alone
(.tangle-solidup.) for blocking IGF1-stimulated AKT
phosphorylation.
FIG. 22. Evaluation of E/I .sup.10Fn3-based binders for inhibition
of cell proliferation in H292 cells. I1-GS10-E5-pegylated
(.quadrature.) was more potent than I1-pegylated alone
(.tangle-solidup.) and E5-pegylated alone (.circle-solid.) had only
weak effects for inhibiting the growth of H292 cells. Assays were
carried out in triplicate. Representative data is shown.
FIG. 23. Evaluation of E/I .sup.10Fn3-based binders for inhibition
of cell proliferation in RH41 cells. I1-GS10-E5-pegylated
(.quadrature.) was slightly more potent than I1-pegylated alone
(.tangle-solidup.) and E5-pegylated alone (.circle-solid.) or
panitumumab (dashed line) had almost no effect for inhibiting the
growth of RH41 cells. Assays were carried out in triplicate.
Representative data is shown.
FIG. 24. Inhibition of ligand stimulated signaling by
.sup.10Fn3-based binders (pegylated). Effect of E/I
.sup.10Fn3-based binder (I1-GS10-E5 pegylated) on receptor
activation and cell signaling in DiFi (Panel A), H292 (Panel B) or
BxPC3 (Panel C) cells. Cells were serum starved and treated for 2
hours with 1 .mu.M .sup.10Fn3-based binders before stimulation with
either EGF, IGF1 or a combination of EGF+IGF1. GAPDH was probed to
illustrate equal loading in all lanes.
FIG. 25. Inhibition of ligand stimulated signaling in H292 cells by
.sup.10Fn3-based binders (unpegylated). Effect of E/I
.sup.10Fn3-based binder (E2-GS10-I1) on receptor activation and
cell signaling in H292 cells. Cells were serum starved and treated
for 2 hours with 1 .mu.M .sup.10Fn3-based binders before
stimulation with either EGF, IGF1 or a combination of EGF+IGF1.
GAPDH was probed to illustrate equal loading in all lanes
FIG. 26. Competition binding studies with E/I .sup.10Fn3-based
binders. A. The EGFR .sup.10Fn3-based binder does not compete for
binding of EGFR antibodies to EGFR. Initial injection of the EGFR
.sup.10Fn3-based binder shows binding to EGFR on the surface of the
chip. A second injection of EGFR .sup.10Fn3-based binder mixed with
an equal amount of cetuximab, panitumumab, or nimotuzumab shows no
competition for binding of antibodies to EGFR by the EGFR
.sup.10Fn3-based binder. B. The E/I .sup.10Fn3-based binder can
bind EGFR and IGF-IR simultaneously. Initial injection of the E/I
.sup.10Fn3-based binder shows binding to EGFR immobilized on the
chip surface. A second injection of the E/I .sup.10Fn3-based binder
soluble IGF-IR shows binding of sIGF-IR to other end of the
immobilized E/I .sup.10Fn3-based binder.
FIG. 27. TGF.alpha. plasma levels 4 hours after last dose of
xenograft studies. Plasma samples taken at the end of treatment
from the BxPC3 (Panel A), GEO (Panel B) and H441 (Panel C)
xenograft studies described in Table 24 were analyzed for
circulating levels of TGF.alpha..
FIG. 28. TGF.alpha. and IGF1 plasma levels in non tumor bearing
nude mice after dosing with I1-GS10-E5 pegylated. Non-tumor bearing
mice were given a single dose of I1-GS10-E5 pegylated
.sup.10Fn3-based binder and analyzed for circulating levels of
TGF.alpha. (Panel A) and IGF1 (Panel B).
FIG. 29. H292 xenograft study using E/I 10Fn3-based binders as
compared to panitumumab. H292 xenografts were either untreated
(.box-solid.) or dosed three times a week with .sup.10Fn3-based
binders formulated in PBS with the individual constructs as
described in the figure or dosed every three days i.p. with
panitumumab at 1 mg/mouse (.largecircle.) or 0.1 mg/mouse
(.quadrature.). Actual doses of .sup.10Fn3-based binders and
panitumumab (.tangle-solidup.) are indicated on the x-axis with the
panitumumab doses closest to the x-axis below the triangles
indicating doses of .sup.10Fn3-based binders. FIG. 29A shows
measurements out to day 43. FIG. 29B shows measurements out to day
27.
FIG. 30. Pharmaokinectic parameters profile of E2-GS10-I1 pegylated
in mice.
FIG. 31. Comparison of half-life at 100 mg/kg and 10 mg/kg IP, and
10 mg/kg and 64 mg/kg SC in various E/I .sup.10Fn3-based
binders.
FIG. 32. Antitumor efficacy of E2-GS10-I1 pegylated in the RH41
model.
FIG. 33. Measurement of pharmacodynamic endpoints in tumors. At the
end of treatment, tumors were removed 4 hours following the final
dose from DiFi xenograft model (panel A) and H292 xenograft model
(panel B) and examined for levels of phospho-EGFR, phospho-IGFR,
total EGFR and total IGFR. Equal amounts of total protein lysate
was loaded into each lane of the gels and blots were also probed
with GAPDH to demonstrate equal loading across all lanes.
FIG. 34. Sequence of anti-EGFR binder 679F09 (SEQ ID NO: 490). Loop
residues which were varied are underlined.
FIG. 35. BC loop Sequence Analysis I. Frequency of amino acids at
each position in the BC loop from EGFR binding sequences. Image
created using WebLogo (Crooks G E, Hon G, Chandonia J M, Brenner S
E. WebLogo: A sequence logo generator. Genome Research,
14:1188-1190, 2004).
FIG. 36. DE loop Sequence Analysis 1. Frequency of amino acids at
each position in the DE loop from EGFR binding sequences (263
unique DE loop sequences analyzed).
FIG. 37. FG loop (10-aa length) Sequence Analysis I. Frequency of
amino acids at each position in the FG loop from EGFR binding
sequences with 10-amino acid long FG loops (228 unique 10-amino
acid long FG loops analyzed).
FIG. 38. FG loop (15-aa length) Sequence Analysis I. Frequency of
amino acids at each position in the FG loop from EGFR binding
sequences with 15-amino acid long FG loops (349 unique 15-amino
acid long FG loops analyzed).
FIG. 39. BC loop Sequence Analysis II. Frequency of amino acids at
each position in the BC loop from all "potent" sequences (85 unique
BC loop sequences analyzed).
FIG. 40. DE loop Sequence Analysis II. Frequency of amino acids at
each position in the DE loop from all "potent" sequences (60 unique
DE loop sequences analyzed).
FIG. 41. FG loop (10-aa length) Sequence Analysis II. Frequency of
amino acids at each position in the FG loop from all "potent"
sequences with 10-amino acid long FG loops (6 unique 10-amino acid
long FG loops analyzed).
FIG. 42. FG loop (15-aa length) Sequence Analysis II. Frequency of
amino acids at each position in the FG loop from all "potent"
sequences with 15-amino acid long FG loops (65 unique 15-amino acid
long FG loops analyzed).
FIG. 43. Table summarizing various characteristics of E/I
.sup.10Fn3-based binders as described in Example 22.
FIG. 44. Table summarizing various pharmacokinetic parameters of
E/I .sup.10Fn3-based binders as described in Example 30.
FIGS. 45A-H. Amino acid sequences of E monomers as described in
Example 32. The BC, DE and FG loops in each sequence are
underlined.
FIG. 46. Alignment of wild-type core sequence (amino acids 9-94 of
SEQ ID NO: 1) with I1 core (SEQ ID NO:65), E1 core (SEQ ID NO:66),
E2 core (SEQ ID NO:67), E3 core (SEQ ID NO:68), E4 core (SEQ ID
NO:108), E5 core (SEQ ID NO:114), E85 core (SEQ ID NO:141), E90
core (SEQ ID NO:156), E96 core (SEQ ID NO:171), E105 core (SEQ ID
NO:186), and E112 core (SEQ ID NO:199). The BC, DE and FG loops in
the wild-type sequences are shown in bold and underlined. The amino
acid residues actually changed in comparison to wild-type for the I
and E cores are shown in bold and underlined.
FIGS. 47A-Q. Nucleic acid sequences of E and I monomers. Unless
otherwise specified, the nucleotide sequences encode a monomer
having an N+10 N-terminal extension, a Ser tail, and a His tag.
FIGS. 48A-G. Nucleic acid sequence of E/I .sup.10Fn3-based binders.
All nucleotide sequences encode an E/I .sup.10Fn3-based binder
having an N+10 N-terminal extension on the first monomer in the
construct and a Cys tail and His tag on the second monomer in the
construct. GS10 is SEQ ID NO: 11; GSGCGS8 is SEQ ID NO: 218; and
GSGC is SEQ ID NO: 489.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
As used herein, the following terms and phrases shall have the
meanings set forth below. Unless defined otherwise, all technical
and scientific terms used herein have the same meaning as commonly
understood to one of ordinary skill in the art.
The singular forms "a," "an," and "the" include plural reference
unless the context clearly dictates otherwise.
The terms "comprise" and "comprising" are used in the inclusive,
open sense, meaning that additional elements may be included.
The term "including" is used to mean "including but not limited
to". "Including" and "including but not limited to" are used
interchangeably.
The term "antibody-like protein" refers to a non-immunoglobulin
protein having an "immunoglobulin-like fold", i.e., comprises about
80-150 amino acid residues that are structurally organized into a
set of beta or beta-like strands, forming beta sheets, where the
beta or beta-like strands are connected by intervening loop
portions. The beta sheets form the stable core of the antibody-like
protein, while creating two "faces" composed of the loops that
connect the beta or beta-like strands. As described herein, these
loops can be varied to create customized ligand binding sites, and
such variations can be generated without disrupting the overall
stability of the protein. An example of such an antibody-like
protein is a "fibronectin-based scaffold protein", by which is
meant a polypeptide based on a fibronectin type III domain (Fn3).
In one aspect, an antibody-like protein is based on a tenth
fibronectin type III domain (.sup.10Fn3).
By a "polypeptide" is meant any sequence of two or more amino
acids, regardless of length, post-translation modification, or
function. "Polypeptide," "peptide," and "protein" are used
interchangeably herein.
"Percent (%) amino acid sequence identity" herein is defined as the
percentage of amino acid residues in a candidate sequence that are
identical with the amino acid residues in a selected sequence,
after aligning the sequences and introducing gaps, if necessary, to
achieve the maximum percent sequence identity, and not considering
any conservative substitutions as part of the sequence identity.
Alignment for purposes of determining percent amino acid sequence
identity can be achieved in various ways that are within the skill
in the art, for instance, using publicly available computer
software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign
(DNASTAR) software. Those skilled in the art can determine
appropriate parameters for measuring alignment, including any
algorithms needed to achieve maximal alignment over the full-length
of the sequences being compared. For purposes herein, however, %
amino acid sequence identity values are obtained as described below
by using the sequence comparison computer program ALIGN-2. The
ALIGN-2 sequence comparison computer program was authored by
Genentech, Inc. has been filed with user documentation in the U.S.
Copyright Office, Washington D.C., 20559, where it is registered
under U.S. Copyright Registration No. TXU510087, and is publicly
available through Genentech, Inc., South San Francisco, Calif. The
ALIGN-2 program should be compiled for use on a UNIX operating
system, preferably digital UNIX V4.0D. All sequence comparison
parameters are set by the ALIGN-2 program and do not vary.
For purposes herein, the % amino acid sequence identity of a given
amino acid sequence A to, with, or against a given amino acid
sequence B (which can alternatively be phrased as a given amino
acid sequence A that has or comprises a certain % amino acid
sequence identity to, with, or against a given amino acid sequence
B) is calculated as follows: 100 times the fraction X/Y where X is
the number of amino acid residues scored as identical matches by
the sequence alignment program ALIGN-2 in that program's alignment
of A and B, and where Y is the total number of amino acid residues
in B. It will be appreciated that where the length of amino acid
sequence A is not equal to the length of amino acid sequence B, the
% amino acid sequence identity of A to B will not equal the % amino
acid sequence identity of B to A.
The term "therapeutically effective amount" refers to an amount of
a drug effective to treat a disease or disorder in a mammal. In the
case of cancer, the therapeutically effective amount of the drug
may reduce the number of cancer cells; reduce the tumor size;
inhibit (i.e., slow to some extent and preferably stop) cancer cell
infiltration into peripheral organs; inhibit (i.e., slow to some
extent and preferably stop) tumor metastasis; inhibit, to some
extent, tumor growth; and/or relieve to some extent one or more of
the symptoms associated with the disorder. To the extent the drug
may prevent growth and/or kill existing cancer cells, it may be
cytostatic and/or cytotoxic. For cancer therapy, efficacy in vivo
can, for example, be measured by assessing the time to disease
progression (TTP) and/or determining the response rates (RR).
The half-life of an amino acid sequence or compound can generally
be defined as the time taken for the serum concentration of the
polypeptide to be reduced by 50% in vivo due to, e.g., degradation
of the sequence or compound and/or clearance or sequestration of
the sequence or compound by natural mechanisms. The half-life can
be determined in any manner known in the art, such as by
pharmacokinetic analysis. See e.g., M Gibaldi & D Perron
"Pharmacokinetics", published by Marcel Dekker, 2nd Rev. edition
(1982).
The term "E/I binder" refers to a bispecific molecule that
comprises an EGFR binding domain and a distinct IGFIR binding
domain. The two domains may be covalently or non-covalently linked.
An exemplary E/I binder is an antibody-like dimer comprising an
EGFR binding .sup.10Fn3 and an IGFIR binding .sup.10Fn3, i.e., an
E/I .sup.10Fn3 based binder.
Overview
The epidermal growth factor receptor (EGFR) and insulin-like growth
factor receptor (IFGR) play key roles in the tumorigenesis of
several types of human cancer. Inhibition of either receptor
effectively reduces tumor growth in preclinical models as well as
clinically. Blocking the EGFR pathway induces switching to the IGFR
pathway to drive growth with in vitro tumor models. Therefore,
blocking both receptors simultaneously may achieve superior
efficacy to blocking either pathway alone by overcoming pathway
switching. In exemplary embodiments, the activity of an E/I binder
is synergistic in comparison to the monomeric components of the E/I
binder.
The specification describes, inter alia, bispecific molecules that
bind EGFR and IGFIR, referred to herein as "E/I binders".
Applicants have discovered that such bispecific molecules inhibit
proliferation of a cancer model cell line with greater potency than
the corresponding. monospecific binders (see e.g., Example 9 and
FIG. 8).
E/I binders will be useful in numerous therapeutic applications,
especially in the treatment of cancer. In addition to therapeutic
applications, E/I binders may be used in any circumstance where it
is desirable to detect EGFR and/or IGFIR.
E/I binders have an EGFR binding domain and a distinct IGFIR
binding domain. Typical binding domains include antibodies;
therefore, bispecific antibodies may be generated to function as
E/I binders. Bispecific antibodies comprising complementary pairs
of V.sub.H and V.sub.L regions are known in the art. These
bispecific antibodies comprise two pairs of V.sub.H and V.sub.L,
each V.sub.H/L pair binding to a single antigen. (see e.g., Hu et
al., Cancer Res. 1996 56:3055-306; Neri et al., J. Mol. Biol. 1995
246:367-373; Atwell et al., Mol. Immunol. 1996 33:1301-1312; and
Carter et al., Protein Sci. 1997 6:781-788). An exemplary
bispecific antibody is a diabody, i.e., a small antibody fragment
with two antigen-binding sites, which fragments comprise a
heavy-chain variable domain connected to a light-chain variable
domain in the same polypeptide chain (Hollinger et al., Proc. Natl.
Acad. Sci. USA 1993 90: 6444-6448).
E/I binders also encompass dimers of ligand binding scaffold
proteins. Scaffold proteins are well described in the literature
and include, e.g., tendamistat, affibody, fibronectin type III
domain, anticalin, tetranectin, and ankyrin. Additional scaffold
proteins that may be used to generate E/I binders are reviewed in
Binz et al., Nature Biotech 23:1257-1268 (2005). Scaffold proteins
are based on a rigid core structure or `framework` that is
important in determining and stabilizing the three-dimensional
structure. In between the fixed or conserved residues of the
scaffold lie variable regions such as loops, surfaces or cavities
that can be randomized to alter ligand binding. A large diversity
of amino acids is provided in the variable regions between the
fixed scaffold residues to provide specific binding to a target
molecule.
An exemplary ligand binding scaffold protein is based on a
fibronectin type III domain (Fn3). Fibronectin is a large protein
which plays essential roles in the formation of extracellular
matrix and cell-cell interactions; it consists of many repeats of
three types (types I, II, and III) of small domains.
Fn3 is small, monomeric, soluble, and stable. It lacks disulfide
bonds and, therefore, is stable under reducing conditions. The
overall structure of Fn3 resembles the immunoglobulin fold. Fn3
domains comprise, in order from N-terminus to C-terminus, a beta or
beta-like strand, A; a loop, AB; a beta or beta-like strand, B; a
loop, BC; a beta or beta-like strand, C; a loop, CD; a beta or
beta-like strand, D; a loop, DE; a beta or beta-like strand, E; a
loop, EF; a beta or beta-like strand, F; a loop, FG; and a beta or
beta-like strand, G. The seven antiparallel .beta.-strands are
arranged as two beta sheets that form a stable core, while creating
two "faces" composed of the loops that connect the beta or
beta-like strands. Loops AB, CD, and EF are located at one face and
loops BC, DE, and FG are located on the opposing face. Any or all
of loops AB, BC, CD, DE, EF and FG may participate in ligand
binding. There are at least 15 different modules of Fn3, and while
the sequence homology between the molecules is low, they all share
a high similarity in tertiary structure.
Adnectins.TM. (Adnexus, a Bristol-Myers Squibb R&D Company) are
ligand binding scaffold proteins based on the tenth fibronectin
type III domain, i.e., the tenth module of Fn3, (.sup.10Fn3). The
amino acid sequence of a naturally occurring human .sup.10Fn3 is
set forth in SEQ ID NO: 1.
TABLE-US-00001 (SEQ ID NO: 1)
VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTV
PGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRT (BC, FG, and DE loops
are emphasized)
In SEQ ID NO:1, the AB loop corresponds to residues 15-16, the BC
loop corresponds to residues 21-30, the CD loop corresponds to
residues 39-45, the DE loop corresponds to residues 51-56, the EF
loop corresponds to residues 60-66, and the FG loop corresponds to
residues 76-87. (Xu et al., Chemistry & Biology 2002
9:933-942). The BC, DE and FG loops align along one face of the
molecule and the AB, CD and EF loops align along the opposite face
of the molecule. In SEQ ID NO: 1, beta strand A corresponds to
residues 9-14, beta strand B corresponds to residues 17-20, beta
strand C corresponds to residues 31-38, beta strand D corresponds
to residues 46-50, beta strand E corresponds to residues 57-59,
beta strand F corresponds to residues 67-75, and beta strand G
corresponds to residues 88-94. The strands are connected to each
other through the corresponding loop, e.g., strands A and B are
connected via loop AB in the formation strand A, loop AB, strand B,
etc. Residues involved in forming the hydrophobic core (the "core
amino acid residues") include the amino acids corresponding to the
following amino acids of SEQ ID NO: 1: L8, V10, A13, L18, I20, W22,
Y32, I34, Y36, F48, V50, A57, I59, L62, Y68, I70, V72, A74, I88,
190 and Y92, wherein the core amino acid residues are represented
by the single letter amino acid code followed by the position at
which they are located within SEQ ID NO: 1. See e.g., Dickinson et
al., J. Mol. Biol. 236: 1079-1092 (1994).
As described above, amino acid residues corresponding to residues
21-30, 51-56, and 76-87 of SEQ ID NO: 1 define the BC, DE and FG
loops, respectively. However, it should be understood that not
every residue within the loop region needs to be modified in order
to achieve a .sup.10Fn3 binder having strong affinity for a desired
target, such as IGF-IR or EGFR. For example, in many of the
examples described herein, only residues corresponding to amino
acids 23-30, 52-55 and 77-86 of SEQ ID NO: 1 were modified to
produce high affinity .sup.10Fn3 binders (see FIG. 46. Accordingly,
in certain embodiments, the BC loop may be defined by amino acids
corresponding to residues 23-30 of SEQ ID NO: 1, the DE loop may be
defined by amino acids corresponding to residues 52-55 of SEQ ID
NO: 1, and the FG loop may be defined by amino acids corresponding
to residues 77-86 of SEQ ID NO: 1.
.sup.10Fn3 are structurally and functionally analogous to
antibodies, specifically the variable region of an antibody. While
.sup.10Fn3 domains may be described as "antibody mimics" or
"antibody-like proteins", they do offer a number of advantages over
conventional antibodies. In particular, they exhibit better folding
and thermostability properties as compared to antibodies, and they
lack disulphide bonds, which are known to impede or prevent proper
folding under certain conditions. Exemplary E/I .sup.10Fn3 based
binders are predominantly monomeric with Tm's averaging
.about.50.degree. C.
The BC, DE, and FG loops of .sup.10Fn3 are analogous to the
complementary determining regions (CDRs) from immunoglobulins.
Alteration of the amino acid sequence in these loop regions changes
the binding specificity of .sup.10Fn3. The protein sequences
outside of the CDR-like loops are analogous to the framework
regions from immunoglobulins and play a role in the structural
conformation of the .sup.10Fn3. Alterations in the framework-like
regions of .sup.10Fn3 are permissible to the extent that the
structural conformation is not so altered as to disrupt ligand
binding. Methods for generating .sup.10Fn3 ligand specific binders
have been described in PCT Publication Nos. WO 00/034787, WO
01/64942, and WO 02/032925, disclosing high affinity TNF.alpha.
binders, PCT Publication No. WO 2008/097497, disclosing high
affinity VEGFR2 binders, and PCT Publication No. WO 2008/066752,
disclosing high affinity IGFIR binders. Additional references
discussing .sup.10Fn3 binders and methods of selecting binders
include PCT Publication Nos. WO 98/056915, WO 02/081497, and WO
2008/031098 and U.S. Publication No. 2003186385.
Antibody-like proteins based on the .sup.10Fn3 scaffold can be
defined generally by the sequence:
VSDVPRDLEVVAATPTSLLI(X).sub.nYYRITYGETGGNSPVQEFTV(X).sub.oATISGLKPGVDYTIT-
V YAV(X).sub.pISINYRT (SEQ ID NO: 32), wherein n is an integer from
1-20, o is an integer from 1-20, and p is an integer from 1-40. The
BC, DE, and FG loops are represented by (X).sub.n, (X).sub.o, and
(X).sub.p, respectively.
.sup.10Fn3 generally begin with the amino acid residue
corresponding to number 1 of SEQ ID NO: 1. However, domains with
amino acid deletions are also encompassed by the invention. In some
embodiments, amino acid residues corresponding to the first eight
amino acids of SEQ ID NO: 1 are deleted. Additional sequences may
also be added to the N- or C-terminus. For example, an additional
MG sequence may be placed at the N-terminus of .sup.10Fn3. The M
will usually be cleaved off, leaving a G at the N-terminus. In some
embodiments, sequences may be placed at the C-terminus of the
.sup.10Fn3 domain, e.g., EIDKPSQ (SEQ ID NO: 9), EIDKPCQ (SEQ ID
NO: 10), EGSGS (SEQ ID NO: 96) or EGSGC (SEQ ID NO: 97).
The non-ligand binding sequences of .sup.10Fn3, i.e., the
".sup.10Fn3 scaffold", may be altered provided that the .sup.10Fn3
retains ligand binding function and/or structural stability. In
some embodiments, one or more of Asp 7, Glu 9, and Asp 23 are
replaced by another amino acid, such as, for example, a
non-negatively charged amino acid residue (e.g., Asn, Lys, etc.).
These mutations have been reported to have the effect of promoting
greater stability of the mutant .sup.10Fn3 at neutral pH as
compared to the wild-type form (See, PCT Publication No. WO
02/04523). A variety of additional alterations in the .sup.10Fn3
scaffold that are either beneficial or neutral have been disclosed.
See, for example, Batori et al., Protein Eng. 2002 15(12):1015-20;
Koide et al., Biochemistry 2001 40(34):10326-33.
The .sup.10Fn3 scaffold may be modified by one or more conservative
substitutions. As many as 5%, 10%, 20% or even 30% or more of the
amino acids in the .sup.10Fn3 scaffold may be altered by a
conservative substitution without substantially altering the
affinity of the .sup.10Fn3 for a ligand. For example, the scaffold
modification preferably reduces the binding affinity of the
.sup.10Fn3 binder for a ligand by less than 100-fold, 50-fold,
25-fold, 10-fold, 5-fold, or 2-fold. It may be that such changes
will alter the immunogenicity of the .sup.10Fn3 in vivo, and where
the immunogenicity is decreased, such changes will be desirable. As
used herein, "conservative substitutions" are residues that are
physically or functionally similar to the corresponding reference
residues. That is, a conservative substitution and its reference
residue have similar size, shape, electric charge, chemical
properties including the ability to form covalent or hydrogen
bonds, or the like. Preferred conservative substitutions are those
fulfilling the criteria defined for an accepted point mutation in
Dayhoff et al., Atlas of Protein Sequence and Structure 5:345-352
(1978 & Supp.). Examples of conservative substitutions are
substitutions within the following groups: (a) valine, glycine; (b)
glycine, alanine; (c) valine, isoleucine, leucine; (d) aspartic
acid, glutamic acid; (e) asparagine, glutamine; (f) serine,
threonine; (g) lysine, arginine, methionine; and (h) phenylalanine,
tyrosine.
E Binders
In one aspect, the disclosure provides antibody-like proteins
comprising an EGFR binding .sup.10Fn3 domain. In certain
embodiments, an EGFR binding .sup.10Fn3 may be provided as part of
a fusion protein or multimer. For example, an EGFR binding
.sup.10Fn3 may be covalently or non-covalently linked to at least a
second .sup.10Fn3 binding domain. The second .sup.10Fn3 binding
domain may bind to EGFR or to a different target. In an exemplary
embodiment, an EGFR binding .sup.10Fn3 may be covalently or
non-covalently linked to an IGF-IR binding .sup.10Fn3.
In exemplary embodiments, the EGFR binding .sup.10Fn3 proteins
described herein bind to EGFR with a K.sub.D of less than 500 nM,
100 nM, 50 nM, 10 nM, 1 nM, 500 pM, 100 pM. 100 pM, 50 pM or 10
pM.
In exemplary embodiments, the BC loop of the EGFR binding
.sup.10Fn3 proteins correspond to amino acids 23-30 of SEQ ID NO:
1, the DE loop of the EGFR binding .sup.10Fn3 proteins correspond
to amino acids 52-55 of SEQ ID NO: 1, and the FG loop of the EGFR
binding .sup.10Fn3 proteins correspond to amino acids 77-86 of SEQ
ID NO: 1.
In one embodiment, the invention provides an EGFR binding
.sup.10Fn3 comprising a BC loop having a YQ at the positions
corresponding to amino acids 29 and 30 of SEQ ID NO: 1.
In another embodiment, the invention provides an EGFR binding
.sup.10Fn3 comprising a BC loop having a YQ at the positions
corresponding to amino acids 29 and 30 of SEQ ID NO: 1 and an FG
loop that is fifteen amino acids in length, e.g., an FG loop that
is extended in length by five amino acids due to an insertion of
five amino acids between residues corresponding to amino acids
77-86 of SEQ ID NO: 1.
In another embodiment, the invention provides an EGFR binding
.sup.10Fn3 comprising a BC loop having a YQ at the positions
corresponding to amino acids 29 and 30 of SEQ ID NO: 1 and a DE
loop having a V, I, L, M or A residue at the position corresponding
to amino acid 54 of SEQ ID NO: 1.
In another embodiment, the invention provides an EGFR binding
.sup.10Fn3 comprising a BC loop having a YQ at the positions
corresponding to amino acids 29 and 30 of SEQ ID NO: 1, a DE loop
having a V, I, L, M or A residue at the position corresponding to
amino acid 54 of SEQ ID NO: 1, and an FG loop that is fifteen amino
acids in length, e.g., an FG loop that is extended in length by
five amino acids due to an insertion of five amino acids between
residues corresponding to amino acids 77-86 of SEQ ID NO: 1.
In another embodiment, the invention provides an EGFR binding
.sup.10Fn3 comprising a BC loop having a YQ at the positions
corresponding to amino acids 29 and 30 of SEQ ID NO: 1 and an FG
loop comprising a D or N at the position corresponding to amino
acid 77 of SEQ ID NO: 1.
In another embodiment, the invention provides an EGFR binding
.sup.10Fn3 comprising a BC loop having a YQ at the positions
corresponding to amino acids 29 and 30 of SEQ ID NO: 1 and an FG
loop (i) that is fifteen amino acids in length, e.g., an FG loop
that is extended in length by five amino acids due to an insertion
of five amino acids between residues corresponding to amino acids
77-86 of SEQ ID NO: 1 and (ii) comprises a D or N at the position
corresponding to amino acid 77 of SEQ ID NO: 1.
In another embodiment, the invention provides an EGFR binding
.sup.10Fn3 comprising a DE loop comprising a V, I, L, M or A
residue at the position corresponding to amino acid 54 of SEQ ID
NO: 1 and an FG loop comprising a D or N at the position
corresponding to amino acid 77 of SEQ ID NO: 1.
In another embodiment, the invention provides an EGFR binding
.sup.10Fn3 comprising a DE loop comprising a V, I, L, M or A
residue at the position corresponding to amino acid 54 of SEQ ID
NO: 1 and an FG loop (i) that is fifteen amino acids in length,
e.g., an FG loop that is extended in length by five amino acids due
to an insertion of five amino acids between residues corresponding
to amino acids 77-86 of SEQ ID NO: 1 and (ii) comprises a D or N at
the position corresponding to amino acid 77 of SEQ ID NO: 1.
In another embodiment, the invention provides an EGFR binding
.sup.10Fn3 comprising a BC loop having a YQ at the positions
corresponding to amino acids 29 and 30 of SEQ ID NO: 1, a DE loop
comprising a V, I, L, M or A residue at the position corresponding
to amino acid 54 of SEQ ID NO: 1, and an FG loop comprising a D or
N at the position corresponding to amino acid 77 of SEQ ID NO:
1.
In another embodiment, the invention provides an EGFR binding
.sup.10Fn3 comprising a BC loop having a YQ at the positions
corresponding to amino acids 29 and 30 of SEQ ID NO: 1, a DE loop
comprising a V, I, L, M or A residue at the position corresponding
to amino acid 54 of SEQ ID NO: 1, and an FG loop (i) that is
fifteen amino acids in length, e.g., an FG loop that is extended in
length by five amino acids due to an insertion of five amino acids
between residues corresponding to amino acids 77-86 of SEQ ID NO: 1
and (ii) comprises a D or N at the position corresponding to amino
acid 77 of SEQ ID NO: 1.
In another embodiment, the invention provides an EGFR binding
.sup.10Fn3 comprising a BC loop comprising the amino acid sequence
XXXXXXYQ, a DE loop comprising the amino acid sequence
XX(V/I/L/M/A)X, and an FG loop comprising the amino acid sequence
(D/N)X.sub.n, wherein X is any amino acid and n is 9-14 amino
acids. In an exemplary embodiment, n is 14 amino acids.
In another embodiment, the invention provides an EGFR binding
.sup.10Fn3 comprising a BC loop corresponding to amino acids 23-30
of SEQ ID NO: 1 comprising the amino acid sequence XXXXXXYQ, a DE
loop corresponding to amino acids 52-55 of SEQ ID NO: 1 comprising
the amino acid sequence XX(V/I/L/M/A)X, and an FG loop
corresponding to amino acids 77-86 of SEQ ID NO: 1 comprising the
amino acid sequence (D/N)X.sub.n, wherein X is any amino acid and n
is 9-14 amino acids. In an exemplary embodiment, n is 14 amino
acids.
In another embodiment, the invention provides an EGFR binding
.sup.10Fn3 comprising a BC loop comprising the amino acid sequence
XXXXXXYQ, a DE loop comprising the amino acid sequence
XX(V/I/L/M/A)X, and an FG loop comprising an amino acid sequence
selected from:
TABLE-US-00002 i. (D/N)(Y/M)(Y/A/M)(Y/H/F)(K/Q/V)(E/P/R)(Y/T/K)
X(E/Y/Q)(Y/G/H); and ii.
D(Y/F/W)(Y/F/K)(N/D/P)(P/H/L)(A/T/V)(T/D/S)
(H/Y/G)(E/P/V)(Y/H)(T/K/I)(Y/F)(H/N/Q)(T/Q/E) (T/S/I);
wherein X is any amino acid.
In another embodiment, the invention provides an EGFR binding
.sup.10Fn3 comprising a BC loop comprising the amino acid sequence
XXXXXXYQ, a DE loop comprising the amino acid sequence
(G/Y/H)(D/M/G)(V/L/I)X, and an FG loop comprising an amino acid
sequence
(D/N)(Y/M)(Y/A/M)(Y/H/F)(K/Q/V)(E/P/R)(Y/T/K)X(E/Y/Q)(Y/G/H),
wherein X is any amino acid.
In another embodiment, the invention provides an EGFR binding
.sup.10Fn3 comprising a BC loop comprising the amino acid sequence
XXXXXXYQ, a DE loop comprising the amino acid sequence
(G/Y/H)(D/M/G)(V/L/I)X, and an FG loop comprising an amino acid
sequence
D(Y/F/W)(Y/F/K)(N/D/P)(P/H/L)(A/T/V)(T/D/S)(H/Y/G)(E/P/V)(Y/H)(T/K/I)(Y/F-
)(H/N/Q)(T/Q/E)(T/S/I), wherein X is any amino acid.
In another embodiment, the invention provides an EGFR binding
.sup.10Fn3 comprising a BC loop comprising the amino acid sequence
XXXXXXYQ, a DE loop comprising the amino acid sequence
XX(V/I/L/M/A)X, and an FG loop comprising an amino acid sequence
selected from:
TABLE-US-00003 (SEQ ID NO: 473) i. DY(A/Y)GKPYXEY; (SEQ ID NO: 474)
ii. DY(A/Y)Y(K/R/Q/T)PYXEY; (SEQ ID NO: 475) iii.
(D/N)Y(A/Y)(Y/F)(K/R/Q/T)EYXE(Y/H); (SEQ ID NO: 476) iv.
DYY(H/Y)X(R/K)X(E/T)YX; (SEQ ID NO: 477) v.
DYY(H/Y)(K/H/Q)(R/K)T(E/T)Y(G/P); (SEQ ID NO: 478) vi.
(D/N)MMHV(E/D)YXEY; (SEQ ID NO: 479) vii. DYMHXXYXEY; and (SEQ ID
NO: 480) viii. D(M/Y)YHX(K/R)X(V/I/L/M)YG;
wherein X is any amino acid.
In another embodiment, the invention provides an EGFR binding
.sup.10Fn3 comprising a BC loop comprising the amino acid sequence
XXXXXXYQ, a DE loop comprising the amino acid sequence
XX(V/I/L/M/A)X, and an FG loop comprising an amino acid sequence
selected from:
TABLE-US-00004 i. D(Y/F)(Y/F)NPXTHEYXYXXX; (SEQ ID NO: 481) ii.
D(Y/F)(Y/F)D(P/L)X(T/S)HXYXYXXX; (SEQ ID NO: 482) and iii.
D(Y/F)(K/R)PHXDGPH(T/I)YXE(S/Y); (SEQ ID NO: 483)
wherein X is any amino acid.
In another embodiment, the invention provides an EGFR binding
.sup.10Fn3 comprising a BC loop comprising the amino acid sequence
XXXXXXYQ, a DE loop comprising the amino acid sequence
XX(V/I/L/M/A)X, and an FG loop comprising the amino acid sequence
(D/N)(M/Y)(M/A/W)(H/F/Y)(V/K)EY(A/Q/R/S/T)E(Y/H/D), wherein X is
any amino acid.
In another embodiment, the invention provides an EGFR binding
.sup.10Fn3 comprising a BC loop comprising the amino acid sequence
XXXXXXYQ, a DE loop comprising the amino acid sequence
XX(V/I/L/M/A)X, and an FG loop comprising the amino acid sequence
D(Y/F/W)(Y/F/K)(N/P/D)(P/H/L)X(T/D/S)(H/G/Y)(E/P/Y)(Y/H)XYXXX,
wherein X is any amino acid.
In various embodiments, the DE loop of the EGFR binding .sup.10Fn3
may comprise the sequence (G/Y/H)(D/M/G)(V/L/I)X.
In another embodiment, the invention provides an EGFR binding
.sup.10Fn3 comprising an FG loop comprising an amino acid sequence
selected from:
TABLE-US-00005 i. D(Y/F)(Y/F)NPXTHEYXYXXX; (SEQ ID NO: 481) ii.
D(Y/F)(Y/F)D(P/L)X(T/S)HXYXYXXX; (SEQ ID NO: 482) and iii.
D(Y/F)(K/R)PHXDGPH(T/I)YXE(S/Y); (SEQ ID NO: 483)
wherein X is any amino acid.
In certain embodiments, the EGFR binding .sup.10Fn3 comprises any
of the consensus sequences provided above, with the proviso that
the EGFR binding .sup.10Fn3 does not comprise one or more of the
following sequences:
TABLE-US-00006 (SEQ ID NO: 484) i.
VSDVPRDLEVVAATPTSLLISWQVPRPMYQRYYRITYGETGGNSPVQEF
TVPGGVRTATISGLKPGVDYTITVYAVTDYMHSEYRQYPISINYRT, and (SEQ ID NO:
485) ii. VSDVPRDLEVVAATPTSLLISWQVPRPMYQYYRITYGETGGNSPVQEF
TVPGGVRTATISGLKPGVDYTITVYAVTDYMHSEYRQYPISINYRT, and (SEQ ID NO:
486) iii. VSDVPRDLEVVAATPTSLLISWQVPRPMYQRYYRITYGETGGNSPVQEF
TVPGGVRTATISGLKPGVDYTITVYAVTDYMHSEYRQYPISINYRTEIDK PCQ.
In certain embodiments, an EGFR binding .sup.10Fn3 comprising one
of the consensus sequences provided above has at least 40%, 50%,
60%, 70%, 75%, or 80% identity to SEQ ID NO: 1. In certain
embodiments, the overall structure of an EGFR binding .sup.10Fn3
comprising one of the consensus sequences provided above resembles
the immunoglobulin fold. In certain embodiment, an EGFR binding
.sup.10Fn3 comprising one of the consensus sequences provided above
further comprises the core amino acid residues of the scaffold. In
certain embodiments, an EGFR binding .sup.10Fn3 comprising one of
the consensus sequences provided above has at least 70%, 75%, 80%,
85%, 90%, 95%, 97%, 98%, or 99% identity to any one of SEQ ID NOs:
5-8, 52, 66-68, 106-108, 112-114, 140-142, 155-157, 170-172, 182,
185-187, 198-200, or 219-327. In certain embodiments, an EGFR
binding .sup.10Fn3 comprising one of the consensus sequences
provided above has at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%,
or 99% identity to the amino acid sequence of amino acid residues
corresponding to E9 of SEQ ID NO: 1 through T94 of SEQ ID NO: 1 of
any one of SEQ ID NOs: 5-8, 52, 66-68, 106-108, 112-114, 140-142,
155-157, 170-172, 182, 185-187, 198-200, or 219-327. In certain
embodiments, the EGFR binding .sup.10Fn3 comprising one of the
consensus sequences provided above comprises a .sup.10Fn3 scaffold
having from has anywhere from 0 to 20, from 0 to 15, from 0 to 10,
from 0 to 8, from 0 to 6, from 0 to 5, from 0 to 4, from 0 to 3,
from 0 to 2, or from 0 to 1 substitutions, conservative
substitutions, deletions or additions relative to the scaffold
amino acids residues of SEQ ID NO: 1.
In certain embodiments, the invention provides an EGFR binding
.sup.10Fn3 comprising a BC loop having the amino acid sequence set
forth in amino acids 23-30, a DE loop having the amino acid
sequence set forth in amino acids 52-55, and an FG loop having the
amino acid sequence set forth in amino acids 77-86 of any one of
SEQ ID NOs: 219-327. In certain embodiments, the invention provides
an EGFR binding .sup.10Fn3 comprising a BC loop having the amino
acid sequence set forth in amino acids 21-30, a DE loop having the
amino acid sequence set forth in amino acids 51-56, and an FG loop
having the amino acid sequence set forth in amino acids 76-87 of
any one of SEQ ID NOs: 219-327. In certain embodiments, the
invention provides an EGFR binding .sup.10Fn3 comprising an amino
acid sequence at least 60%, 75%, 80%, 85%, 90%, 95%, or 98%
identical to any one of SEQ ID NOs: 219-327.
In one embodiment, an antibody-like protein is provided comprising
a tenth fibronectin type III domain (.sup.10Fn3) that binds EGFR
with a K.sub.D of less than 500 nM and comprises a BC loop having
the amino acid sequence set forth in amino acids 21-30 of SEQ ID
NO: 5, a DE loop having the amino acid sequence set forth in amino
acids 51-56 of SEQ ID NO: 5, and an FG loop having the amino acid
sequence set forth in amino acids 76-92 of SEQ ID NO: 5. In some
embodiments, the EGFR binding .sup.10Fn3 comprises a BC loop having
the amino acid sequence X.sub.gDSGRGSYQX.sub.h (SEQ ID NO: 40), a
DE loop having the amino acid sequence X.sub.iGPVHX.sub.j (SEQ ID
NO: 42), and an FG loop having the amino acid sequence
X.sub.kDHKPHADGPHTYHEX.sub.l (SEQ ID NO: 44); wherein X is any
amino acid and g, h, i, j, k, and l are integers independently
selected from 0 to 5. In some embodiments, the EGFR binding
.sup.10Fn3 comprises a BC loop having the amino acid sequence
SWDSGRGSYQ (SEQ ID NO: 39), a DE loop having the amino acid
sequence PGPVHT (SEQ ID NO: 41), and an FG loop having the amino
acid sequence TDHKPHADGPHTYHESP (SEQ ID NO: 43). In some
embodiments, the EGFR binding .sup.10Fn3 has an amino acid sequence
at least 80, 90, 95, or 100% identical to SEQ ID NOs: 5 or 6.
In one embodiment, an antibody-like protein is provided comprising
a tenth fibronectin type III domain (.sup.10Fn3) that binds EGFR
with a K.sub.D of less than 500 nM and comprises a BC loop having
the amino acid sequence set forth in amino acids 21-30 of SEQ ID
NO: 7, a DE loop having the amino acid sequence set forth in amino
acids 51-56 of SEQ ID NO: 7, and an FG loop having the amino acid
sequence set forth in amino acids 76-87 of SEQ ID NO: 7. In some
embodiments, the EGFR binding .sup.10Fn3 comprises a BC loop having
the amino acid sequence X.sub.mVAGAEDYQX.sub.n (SEQ ID NO: 34), a
DE loop having the amino acid sequence X.sub.oHDLVX.sub.p (SEQ ID
NO: 36), and an FG loop having the amino acid sequence
X.sub.qDMMHVEYTEHX.sub.r (SEQ ID NO: 38); wherein X is any amino
acid and m, n, o, p, q, and r are integers independently selected
from 0 to 5. In some embodiments, the EGFR binding .sup.10Fn3
comprises a BC loop having the amino acid sequence SWVAGAEDYQ (SEQ
ID NO: 33), a DE loop having the amino acid sequence PHDLVT (SEQ ID
NO: 35), and an FG loop having the amino acid sequence TDMMHVEYTEHP
(SEQ ID NO: 37). In some embodiments, the EGFR binding .sup.10Fn3
has an amino acid sequence at least 80, 90, 95, or 100% identical
to SEQ ID NO: 7 or 8.
In one embodiment, an antibody-like protein is provided comprising
a tenth fibronectin type III domain (.sup.10Fn3) that binds EGFR
with a K.sub.D of less than 500 nM and comprises a BC loop having
the amino acid sequence set forth in amino acids 23-30 of SEQ ID
NO: 82, a DE loop having the amino acid sequence set forth in amino
acids 51-55 of SEQ ID NO: 82, and an FG loop having the amino acid
sequence set forth in amino acids 76-86 of SEQ ID NO: 82. In some
embodiments, the EGFR binding .sup.10Fn3 comprises a BC loop having
the amino acid sequence X.sub.sLPGKLRYQX.sub.t (SEQ ID NO: 60), a
DE loop having the amino acid sequence X.sub.uHDLRX.sub.w (SEQ ID
NO: 62), and an FG loop having the amino acid sequence
X.sub.yNMMHVEYSEYX.sub.z (SEQ ID NO: 64); wherein X is any amino
acid and s, t, u, w, y and z are integers independently selected
from 0 to 5. In some embodiments, the EGFR binding .sup.10Fn3
comprises a BC loop having the amino acid sequence LPGKLRYQ
(residues 3-13 of SEQ ID NO: 59), a DE loop having the amino acid
sequence PHDLR (residues 1-5 of SEQ ID NO: 61), and an FG loop
having the amino acid sequence TNMMHVEYSEY (residues 1-11 of SEQ ID
NO: 63). In some embodiments, the EGFR binding .sup.10Fn3 has an
amino acid sequence at least 80, 90, 95, or 100% identical to SEQ
ID NO: 52 or 82.
In one embodiment, an antibody-like protein is provided comprising
a tenth fibronectin type III domain (.sup.10Fn3) that binds EGFR
with a K.sub.D of less than 500 nM and comprises a BC loop having
the amino acid sequence set forth in amino acids 23-30 of SEQ ID
NO: 106, a DE loop having the amino acid sequence set forth in
amino acids 51-55 of SEQ ID NO: 106, and an FG loop having the
amino acid sequence set forth in amino acids 76-86 of SEQ ID NO:
106. In some embodiments, the EGFR binding .sup.10Fn3 comprises a
BC loop having the amino acid sequence X.sub.gHERDGSRQX.sub.h (SEQ
ID NO: 134), a DE loop having the amino acid sequence
X.sub.iGGVRX.sub.j (SEQ ID NO: 135), and an FG loop having the
amino acid sequence X.sub.kDYFNPTTHEYIYQTTX.sub.l (SEQ ID NO: 136);
wherein X is any amino acid and g, h, i, j, k and l are integers
independently selected from 0 to 5. In some embodiments, the EGFR
binding .sup.10Fn3 comprises a BC loop having the amino acid
sequence SWHERDGSRQ (SEQ ID NO: 109), a DE loop having the amino
acid sequence PGGVRT (SEQ ID NO: 110), and an FG loop having the
amino acid sequence TDYFNPTTHEYIYQTTP (SEQ ID NO: 111). In some
embodiments, the EGFR binding .sup.10Fn3 has an amino acid sequence
at least 80, 90, 95, or 100% identical to any one of SEQ ID NOs:
106-108.
In one embodiment, an antibody-like protein is provided comprising
a tenth fibronectin type III domain (.sup.10Fn3) that binds EGFR
with a K.sub.D of less than 500 nM and comprises a BC loop having
the amino acid sequence set forth in amino acids 23-30 of SEQ ID
NO: 112, a DE loop having the amino acid sequence set forth in
amino acids 51-55 of SEQ ID NO: 112, and an FG loop having the
amino acid sequence set forth in amino acids 76-86 of SEQ ID NO:
112. In some embodiments, the EGFR binding .sup.10Fn3 comprises a
BC loop having the amino acid sequence X.sub.gWAPVDRYQX.sub.h (SEQ
ID NO: 137), a DE loop having the amino acid sequence
X.sub.iRDVYX.sub.j (SEQ ID NO: 138), and an FG loop having the
amino acid sequence X.sub.kDYKPHADGPHTYHESX.sub.l (SEQ ID NO: 139);
wherein X is any amino acid and g, h, i, j, k and l are integers
independently selected from 0 to 5. In some embodiments, the EGFR
binding .sup.10Fn3 comprises a BC loop having the amino acid
sequence SWWAPVDRYQ (SEQ ID NO: 115), a DE loop having the amino
acid sequence PRDVYT (SEQ ID NO: 116), and an FG loop having the
amino acid sequence TDYKPHADGPHTYHESP (SEQ ID NO: 117). In some
embodiments, the EGFR binding .sup.10Fn3 has an amino acid sequence
at least 80, 90, 95, or 100% identical to any one of SEQ ID NOs:
112-114.
In one embodiment, an antibody-like protein is provided comprising
a tenth fibronectin type III domain (.sup.10Fn3) that binds EGFR
with a K.sub.D of less than 500 nM and comprises a BC loop having
the amino acid sequence set forth in amino acids 13-22 of SEQ ID
NO: 141, a DE loop having the amino acid sequence set forth in
amino acids 43-48 of SEQ ID NO: 141, and an FG loop having the
amino acid sequence set forth in amino acids 68-84 of SEQ ID NO:
141. In some embodiments, the EGFR binding .sup.10Fn3 comprises a
BC loop having the amino acid sequence X.sub.gTQGSTHYQX.sub.h (SEQ
ID NO: 146), a DE loop having the amino acid sequence
X.sub.iGMVYX.sub.j (SEQ ID NO: 147), and an FG loop having the
amino acid sequence X.sub.kDYFDRSTHEYKYRTTX.sub.l (SEQ ID NO: 148);
wherein X is any amino acid and g, h, i, j, k and l are integers
independently selected from 0 to 5. In some embodiments, the EGFR
binding .sup.10Fn3 comprises a BC loop having the amino acid
sequence SWTQGSTHYQ (SEQ ID NO: 143), a DE loop having the amino
acid sequence PGMVYT (SEQ ID NO: 144), and an FG loop having the
amino acid sequence TDYFDRSTHEYKYRTTP (SEQ ID NO: 145). In some
embodiments, the EGFR binding .sup.10Fn3 has an amino acid sequence
at least 80, 90, 95, or 100% identical to any one of SEQ ID NOs:
140-142.
In one embodiment, an antibody-like protein is provided comprising
a tenth fibronectin type III domain (.sup.10Fn3) that binds EGFR
with a K.sub.D of less than 500 nM and comprises a BC loop having
the amino acid sequence set forth in amino acids 13-22 of SEQ ID
NO: 156, a DE loop having the amino acid sequence set forth in
amino acids 43-48 of SEQ ID NO: 156, and an FG loop having the
amino acid sequence set forth in amino acids 68-84 of SEQ ID NO:
156. In some embodiments, the EGFR binding .sup.10Fn3 comprises a
BC loop having the amino acid sequence X.sub.gYWEGLPYQX.sub.h (SEQ
ID NO: 161), a DE loop having the amino acid sequence
X.sub.iRDVNX.sub.j (SEQ ID NO: 162), and an FG loop having the
amino acid sequence X.sub.kDWYNPDTHEYIYHTIX.sub.l (SEQ ID NO: 163);
wherein X is any amino acid and g, h, i, j, k and l are integers
independently selected from 0 to 5. In some embodiments, the EGFR
binding .sup.10Fn3 comprises a BC loop having the amino acid
sequence SWYWEGLPYQ (SEQ ID NO: 158), a DE loop having the amino
acid sequence PRDVNT (SEQ ID NO: 159), and an FG loop having the
amino acid sequence TDWYNPDTHEYIYHTIP (SEQ ID NO: 160). In some
embodiments, the EGFR binding .sup.10Fn3 has an amino acid sequence
at least 80, 90, 95, or 100% identical to any one of SEQ ID NOs:
155-157.
In one embodiment, an antibody-like protein is provided comprising
a tenth fibronectin type III domain (.sup.10Fn3) that binds EGFR
with a K.sub.D of less than 500 nM and comprises a BC loop having
the amino acid sequence set forth in amino acids 13-22 of SEQ ID
NO: 171, a DE loop having the amino acid sequence set forth in
amino acids 43-48 of SEQ ID NO: 171, and an FG loop having the
amino acid sequence set forth in amino acids 68-84 of SEQ ID NO:
171. In some embodiments, the EGFR binding .sup.10Fn3 comprises a
BC loop having the amino acid sequence X.sub.gASNRGTYQX.sub.h (SEQ
ID NO: 176), a DE loop having the amino acid sequence
X.sub.iGGVSX.sub.j (SEQ ID NO: 177), and an FG loop having the
amino acid sequence X.sub.kDAFNPTTHEYNYFTTX.sub.l (SEQ ID NO: 178);
wherein X is any amino acid and g, h, i, j, k and l are integers
independently selected from 0 to 5. In some embodiments, the EGFR
binding .sup.10Fn3 comprises a BC loop having the amino acid
sequence SWASNRGTYQ (SEQ ID NO: 173), a DE loop having the amino
acid sequence PGGVST (SEQ ID NO: 174), and an FG loop having the
amino acid sequence TDAFNPTTHEYNYFTTP (SEQ ID NO: 175). In some
embodiments, the EGFR binding .sup.10Fn3 has an amino acid sequence
at least 80, 90, 95, or 100% identical to any one of SEQ ID NOs:
170-172.
In one embodiment, an antibody-like protein is provided comprising
a tenth fibronectin type III domain (.sup.10Fn3) that binds EGFR
with a K.sub.D of less than 500 nM and comprises a BC loop having
the amino acid sequence set forth in amino acids 13-22 of SEQ ID
NO: 186, a DE loop having the amino acid sequence set forth in
amino acids 43-48 of SEQ ID NO: 186, and an FG loop having the
amino acid sequence set forth in amino acids 68-84 of SEQ ID NO:
186. In some embodiments, the EGFR binding .sup.10Fn3 comprises a
BC loop having the amino acid sequence X.sub.gDAPTSRYQX.sub.h (SEQ
ID NO: 190), a DE loop having the amino acid sequence
X.sub.iGGLSX.sub.j (SEQ ID NO: 191), and an FG loop having the
amino acid sequence X.sub.kDYKPHADGPHTYHESX.sub.l (SEQ ID NO: 139);
wherein X is any amino acid and g, h, i, j, k and l are integers
independently selected from 0 to 5. In some embodiments, the EGFR
binding .sup.10Fn3 comprises a BC loop having the amino acid
sequence SWDAPTSRYQ (SEQ ID NO: 188), a DE loop having the amino
acid sequence PGGLST (SEQ ID NO: 189), and an FG loop having the
amino acid sequence TDYKPHADGPHTYHESP (SEQ ID NO: 117). In some
embodiments, the EGFR binding .sup.10Fn3 has an amino acid sequence
at least 80, 90, 95, or 100% identical to any one of SEQ ID NOs:
185-187.
In one embodiment, an antibody-like protein is provided comprising
a tenth fibronectin type III domain (.sup.10Fn3) that binds EGFR
with a K.sub.D of less than 500 nM and comprises a BC loop having
the amino acid sequence set forth in amino acids 13-22 of SEQ ID
NO: 199, a DE loop having the amino acid sequence set forth in
amino acids 43-48 of SEQ ID NO: 199, and an FG loop having the
amino acid sequence set forth in amino acids 68-84 of SEQ ID NO:
199. In some embodiments, the EGFR binding .sup.10Fn3 comprises a
BC loop having the amino acid sequence X.sub.gDAGAVTYQX.sub.h (SEQ
ID NO: 203), a DE loop having the amino acid sequence
X.sub.iGGVRX.sub.j (SEQ ID NO: 135), and an FG loop having the
amino acid sequence X.sub.kDYKPHADGPHTYHEYX.sub.l (SEQ ID NO: 204);
wherein X is any amino acid and g, h, i, j, k and l are integers
independently selected from 0 to 5. In some embodiments, the EGFR
binding .sup.10Fn3 comprises a BC loop having the amino acid
sequence SWDAGAVTYQ (SEQ ID NO: 201), a DE loop having the amino
acid sequence PGGVRT (SEQ ID NO: 110), and an FG loop having the
amino acid sequence TDYKPHADGPHTYHEYP (SEQ ID NO: 202). In some
embodiments, the EGFR binding .sup.10Fn3 has an amino acid sequence
at least 80, 90, 95, or 100% identical to any one of SEQ ID NOs:
198-200.
In certain embodiments, an EGFR binding .sup.10Fn3 domain is
covalently or non-covalently linked to an EGF-IR binding .sup.10Fn3
domain. In exemplary embodiments, the IGF-IR binding .sup.10Fn3 may
comprise a BC loop having the amino acid sequence set forth in
amino acids 21-30 of SEQ ID NO: 3, a DE loop having the amino acid
sequence set forth in amino acids 51-56 of SEQ ID NO: 3, and an FG
loop having the amino acid sequence set forth in amino acids 76-83
of SEQ ID NO: 3. In some embodiments, the IGF-IR binding .sup.10Fn3
comprises a BC loop having the amino acid sequence
X.sub.aSARLKVAX.sub.b (SEQ ID NO: 46), a DE loop having the amino
acid sequence X.sub.cKNVYX.sub.d (SEQ ID NO: 48), and an FG loop
having the amino acid sequence X.sub.eRFRDYQX.sub.f (SEQ ID NO:
50), wherein X is any amino acid and a, b, c, d, e, f, g, h, i, j,
k, and l are integers independently selected from 0 to 5, or
wherein a is 2 and b-f are 1, or wherein a-f are zero. In some
embodiments, the IGF-IR binding .sup.10Fn3 has an amino acid
sequence at least 80, 90, 95, 98, 99, or 100% identical to SEQ ID
NO: 3. In certain embodiments, the IGF-IR binding .sup.10Fn3
comprises a .sup.10Fn3 scaffold having from has anywhere from 0 to
20, from 0 to 15, from 0 to 10, from 0 to 8, from 0 to 6, from 0 to
5, from 0 to 4, from 0 to 3, from 0 to 2, or from 0 to 1
substitutions, conservative substitutions, deletions or additions
relative to the scaffold amino acid residues of SEQ ID NO: 1. In
certain embodiments, the IGF-IR binding .sup.10Fn3 has anywhere
from 0 to 15, from 0 to 10, from 0 to 8, from 0 to 6, from 0 to 5,
from 0 to 4, from 0 to 3, from 0 to 2, or from 0 to 1
substitutions, conservative substitutions, deletions or additions
relative to the corresponding loop sequences of SEQ ID NO: 3.
.sup.10Fn3 E/I Binders
One aspect of the disclosure provides E/I binders constructed from
antibody-like protein multimers. In some embodiments, an
antibody-like protein multimer comprises at least one EGFR binding
.sup.10Fn3 covalently or non-covalently linked to at least one
IGFIR binding .sup.10Fn3. In certain embodiments, the E/I binders
described herein may be constructed as a single polypeptide chain
wherein the E and I subunits may be in either orientation, e.g.,
from N-terminus to C-terminus, in the E-I orientation or in the I-E
orientation.
The disclosure relates, in part, to the surprising discovery that
multiple .sup.10Fn3 joined via a polypeptide linker correctly fold
independently of each other, retain high affinity binding, and that
each of the domains retains its functional properties (see e.g.,
Examples 5-10). Additionally, these E/I .sup.10Fn3 based binders
demonstrate desirable biophysical properties such as low
aggregation and high melting temperature (T.sub.m) (see e.g.,
Example 4). The Examples characterize a variety of E/I .sup.10Fn3
based binders. An exemplary IGFIR binding .sup.10Fn3 is set forth
in SEQ ID NO: 4. Exemplary EGFR binding .sup.10Fn3 are set forth in
SEQ ID NOs: 6, 8, 52, 107, 113, 140, 155, 170, 185 and 198.
In some embodiments, an E/I binder comprises an EGFR binding
.sup.10Fn3 and an IGFIR binding .sup.10Fn3, independently having an
amino acid sequence at least 40, 50, 60, 70, or 80% identical to
the human .sup.10Fn3 domain, shown in SEQ ID NO: 1. Much of the
variability will generally occur in one or more of the loops.
In some embodiments, an E/I binder comprises an EGFR binding
.sup.10Fn3 and an IGFIR binding .sup.10Fn3, independently having an
amino acid sequence at least 70, 80, 85, 90, 95, 98, or 100%
identical to SEQ ID NO: 32, wherein n is an integer from 1-20, o is
an integer from 1-20, and p is an integer from 1-40. In some
embodiments, n is an integer from 8-12, o is an integer from 4-8,
and p is an integer from 4-28. In some embodiments, n is 10, o is
6, and p is 12.
In some embodiments, the disclosure provides multimers of
.sup.10Fn3 having at least one loop selected from loop BC, DE, and
FG with an altered amino acid sequence relative to the sequence of
the corresponding loop of the human .sup.10Fn3. By "altered" is
meant one or more amino acid sequence alterations relative to a
template sequence (corresponding human fibronectin domain) and
includes amino acid additions, deletions, and substitutions.
Altering an amino acid sequence may be accomplished through
intentional, blind, or spontaneous sequence variation, generally of
a nucleic acid coding sequence, and may occur by any technique, for
example, PCR, error-prone PCR, or chemical DNA synthesis. In some
embodiments, an amino acid sequence is altered by substituting with
or adding naturally occurring amino acids.
In some embodiments, one or more loops selected from BC, DE, and FG
may be extended or shortened in length relative to the
corresponding human fibronectin loop. In particular, the FG loop of
the human .sup.10Fn3 is 12 residues long, whereas the corresponding
loop in antibody heavy chains ranges from 4-28 residues. To
optimize antigen binding, therefore, the length of the FG loop of
.sup.10Fn3 may be altered in length as well as in sequence to
obtain the greatest possible flexibility and affinity in antigen
binding.
In some embodiments of the .sup.10Fn3 molecules, the altered BC
loop has up to 10 amino acid substitutions, up to 9 amino acid
deletions, up to 10 amino acid insertions, or a combination of
substitutions and deletions or insertions. In some embodiments, the
altered DE loop has up to 6 amino acid substitutions, up to 5 amino
acid deletions, up to 14 amino acid insertions or a combination of
substitutions and deletions or insertions. In some embodiments, the
FG loop has up to 12 amino acid substitutions, up to 11 amino acid
deletions, up to 28 amino acid insertions or a combination of
substitutions and deletions or insertions.
Naturally occurring .sup.10Fn3 comprises an
"arginine-glycine-aspartic acid" (RGD) integrin-binding motif in
the FG loop. Preferred multimers of .sup.10Fn3 lack an RGD
integrin-binding motif.
In some embodiments, an E/I binder is an antibody-like protein
dimer comprising a) an EGFR binding .sup.10Fn3 comprising a BC loop
having the amino acid sequence set forth in amino acids 21-30 of
SEQ ID NO: 5, a DE loop having the amino acid sequence set forth in
amino acids 51-56 of SEQ ID NO: 5, and an FG loop having the amino
acid sequence set forth in amino acids 76-92 of SEQ ID NO: 5;
covalently or non-covalently linked to b) an IGFIR binding
.sup.10Fn3 comprising a BC loop having the amino acid sequence set
forth in amino acids 21-30 of SEQ ID NO: 3, a DE loop having the
amino acid sequence set forth in amino acids 51-56 of SEQ ID NO: 3,
and an FG loop having the amino acid sequence set forth in amino
acids 76-83 of SEQ ID NO: 3. In some embodiments, the EGFR binding
.sup.10Fn3 has an amino acid sequence at least 80, 90, 95, 98, 99,
or 100% identical to SEQ ID NO: 5. In some embodiments, the IGFIR
binding .sup.10Fn3 has an amino acid sequence at least 80, 90, 95,
98, 99, or 100% identical to SEQ ID NO: 3. In some embodiments, the
E/I binder comprises an amino acid sequence at least 80, 85, 90,
95, 98, 99, or 100% identical to SEQ ID NOs: 20, 21, 23, 24, 90,
92, 101 or 103.
In some embodiments, an E/I binder is an antibody-like protein
dimer comprising a) an EGFR binding .sup.10Fn3 comprising a BC loop
having the amino acid sequence set forth in amino acids 21-30 of
SEQ ID NO: 7, a DE loop having the amino acid sequence set forth in
amino acids 51-56 of SEQ ID NO: 7, and an FG loop having the amino
acid sequence set forth in amino acids 76-87 of SEQ ID NO: 7;
covalently or non-covalently linked to b) an IGFIR binding
.sup.10Fn3 comprising a BC loop having the amino acid sequence set
forth in amino acids 21-30 of SEQ ID NO: 3, a DE loop having the
amino acid sequence set forth in amino acids 51-56 of SEQ ID NO: 3,
and an FG loop having the amino acid sequence set forth in amino
acids 76-83 of SEQ ID NO: 3. In some embodiments, the EGFR binding
.sup.10Fn3 has an amino acid sequence at least 80, 90, 95, 98, or
100% identical to SEQ ID NO: 7. In some embodiments, the IGFIR
binding .sup.10Fn3 has an amino acid sequence at least 80, 90, 95,
98, or 100% identical to SEQ ID NO: 3. In some embodiments, the E/I
binder comprises an amino acid sequence at least 80, 85, 90, 95,
98, or 100% identical to SEQ ID NOs: 26, 27, 29, 30, 89, 91, 100 or
102.
In some embodiments, an E/I binder is an antibody-like protein
dimer comprising a) an EGFR binding .sup.10Fn3 comprising a BC loop
having the amino acid sequence set forth in amino acids 21-30 of
SEQ ID NO: 82, a DE loop having the amino acid sequence set forth
in amino acids 51-56 of SEQ ID NO: 82, and an FG loop having the
amino acid sequence set forth in amino acids 76-87 of SEQ ID NO:
82; covalently or non-covalently linked to b) an IGFIR binding
.sup.10Fn3 comprising a BC loop having the amino acid sequence set
forth in amino acids 21-30 of SEQ ID NO: 3, a DE loop having the
amino acid sequence set forth in amino acids 51-56 of SEQ ID NO: 3,
and an FG loop having the amino acid sequence set forth in amino
acids 76-83 of SEQ ID NO: 3. In some embodiments, the EGFR binding
.sup.10Fn3 has an amino acid sequence at least 80, 90, 95, 98, or
100% identical to SEQ ID NO: 82. In some embodiments, the IGFIR
binding .sup.10Fn3 has an amino acid sequence at least 80, 90, 95,
98, or 100% identical to SEQ ID NO: 3. In some embodiments, the E/I
binder comprises an amino acid sequence at least 80, 85, 90, 95,
98, or 100% identical to SEQ ID NOs: 53, 54, 87, 88, 98, 99, 104 or
105.
In some embodiments, an E/I binder is an antibody-like protein
dimer comprising a) an EGFR binding .sup.10Fn3 comprising a BC loop
having the amino acid sequence set forth in amino acids 21-30 of
SEQ ID NO: 106, a DE loop having the amino acid sequence set forth
in amino acids 51-56 of SEQ ID NO: 106, and an FG loop having the
amino acid sequence set forth in amino acids 76-92 of SEQ ID NO:
106; covalently or non-covalently linked to b) an IGFIR binding
.sup.10Fn3 comprising a BC loop having the amino acid sequence set
forth in amino acids 21-30 of SEQ ID NO: 3, a DE loop having the
amino acid sequence set forth in amino acids 51-56 of SEQ ID NO: 3,
and an FG loop having the amino acid sequence set forth in amino
acids 76-83 of SEQ ID NO: 3. In some embodiments, the EGFR binding
.sup.10Fn3 has an amino acid sequence at least 80, 90, 95, 98, or
100% identical to SEQ ID NO: 106. In some embodiments, the IGFIR
binding .sup.10Fn3 has an amino acid sequence at least 80, 90, 95,
98, or 100% identical to SEQ ID NO: 3. In some embodiments, the E/I
binder comprises an amino acid sequence at least 80, 85, 90, 95,
98, or 100% identical to SEQ ID NOs: 118-125.
In some embodiments, an E/I binder is an antibody-like protein
dimer comprising a) an EGFR binding .sup.10Fn3 comprising a BC loop
having the amino acid sequence set forth in amino acids 21-30 of
SEQ ID NO: 112, a DE loop having the amino acid sequence set forth
in amino acids 51-56 of SEQ ID NO: 112, and an FG loop having the
amino acid sequence set forth in amino acids 76-92 of SEQ ID NO:
112; covalently or non-covalently linked to b) an IGFIR binding
.sup.10Fn3 comprising a BC loop having the amino acid sequence set
forth in amino acids 21-30 of SEQ ID NO: 3, a DE loop having the
amino acid sequence set forth in amino acids 51-56 of SEQ ID NO: 3,
and an FG loop having the amino acid sequence set forth in amino
acids 76-83 of SEQ ID NO: 3. In some embodiments, the EGFR binding
.sup.10Fn3 has an amino acid sequence at least 80, 90, 95, 98, or
100% identical to SEQ ID NO: 112. In some embodiments, the IGFIR
binding .sup.10Fn3 has an amino acid sequence at least 80, 90, 95,
98, or 100% identical to SEQ ID NO: 3. In some embodiments, the E/I
binder comprises an amino acid sequence at least 80, 85, 90, 95,
98, or 100% identical to SEQ ID NOs: 126-133.
In some embodiments, an E/I binder is an antibody-like protein
dimer comprising a) an EGFR binding .sup.10Fn3 comprising a BC loop
having the amino acid sequence set forth in amino acids 13-22 of
SEQ ID NO: 141, a DE loop having the amino acid sequence set forth
in amino acids 43-48 of SEQ ID NO: 141, and an FG loop having the
amino acid sequence set forth in amino acids 68-84 of SEQ ID NO:
141; covalently or non-covalently linked to b) an IGFIR binding
.sup.10Fn3 comprising a BC loop having the amino acid sequence set
forth in amino acids 21-30 of SEQ ID NO: 3, a DE loop having the
amino acid sequence set forth in amino acids 51-56 of SEQ ID NO: 3,
and an FG loop having the amino acid sequence set forth in amino
acids 76-83 of SEQ ID NO: 3. In some embodiments, the EGFR binding
.sup.10Fn3 has an amino acid sequence at least 80, 90, 95, 98, or
100% identical to SEQ ID NO: 140, 141, 142 or 300. In some
embodiments, the IGFIR binding .sup.10Fn3 has an amino acid
sequence at least 80, 90, 95, 98, or 100% identical to SEQ ID NO:
3. In some embodiments, the E/I binder comprises an amino acid
sequence at least 80, 85, 90, 95, 98, or 100% identical to SEQ ID
NOs: 149-154.
In some embodiments, an E/I binder is an antibody-like protein
dimer comprising a) an EGFR binding .sup.10Fn3 comprising a BC loop
having the amino acid sequence set forth in amino acids 13-22 of
SEQ ID NO: 156, a DE loop having the amino acid sequence set forth
in amino acids 43-48 of SEQ ID NO: 156, and an FG loop having the
amino acid sequence set forth in amino acids 68-84 of SEQ ID NO:
156; covalently or non-covalently linked to b) an IGFIR binding
.sup.10Fn3 comprising a BC loop having the amino acid sequence set
forth in amino acids 21-30 of SEQ ID NO: 3, a DE loop having the
amino acid sequence set forth in amino acids 51-56 of SEQ ID NO: 3,
and an FG loop having the amino acid sequence set forth in amino
acids 76-83 of SEQ ID NO: 3. In some embodiments, the EGFR binding
.sup.10Fn3 has an amino acid sequence at least 80, 90, 95, 98, or
100% identical to SEQ ID NO: 155, 156, 157 or 305. In some
embodiments, the IGFIR binding .sup.10Fn3 has an amino acid
sequence at least 80, 90, 95, 98, or 100% identical to SEQ ID NO:
3. In some embodiments, the E/I binder comprises an amino acid
sequence at least 80, 85, 90, 95, 98, or 100% identical to SEQ ID
NOs: 158-166.
In some embodiments, an E/I binder is an antibody-like protein
dimer comprising a) an EGFR binding .sup.10Fn3 comprising a BC loop
having the amino acid sequence set forth in amino acids 13-22 of
SEQ ID NO: 171, a DE loop having the amino acid sequence set forth
in amino acids 43-48 of SEQ ID NO: 171, and an FG loop having the
amino acid sequence set forth in amino acids 68-84 of SEQ ID NO:
171; covalently or non-covalently linked to b) an IGFIR binding
.sup.10Fn3 comprising a BC loop having the amino acid sequence set
forth in amino acids 21-30 of SEQ ID NO: 3, a DE loop having the
amino acid sequence set forth in amino acids 51-56 of SEQ ID NO: 3,
and an FG loop having the amino acid sequence set forth in amino
acids 76-83 of SEQ ID NO: 3. In some embodiments, the EGFR binding
.sup.10Fn3 has an amino acid sequence at least 80, 90, 95, 98, or
100% identical to SEQ ID NO: 170, 171, 172 or 311. In some
embodiments, the IGFIR binding .sup.10Fn3 has an amino acid
sequence at least 80, 90, 95, 98, or 100% identical to SEQ ID NO:
3. In some embodiments, the E/I binder comprises an amino acid
sequence at least 80, 85, 90, 95, 98, or 100% identical to SEQ ID
NOs: 179-184.
In some embodiments, an E/I binder is an antibody-like protein
dimer comprising a) an EGFR binding .sup.10Fn3 comprising a BC loop
having the amino acid sequence set forth in amino acids 13-22 of
SEQ ID NO: 186, a DE loop having the amino acid sequence set forth
in amino acids 43-48 of SEQ ID NO: 186, and an FG loop having the
amino acid sequence set forth in amino acids 68-84 of SEQ ID NO:
186; covalently or non-covalently linked to b) an IGFIR binding
.sup.10Fn3 comprising a BC loop having the amino acid sequence set
forth in amino acids 21-30 of SEQ ID NO: 3, a DE loop having the
amino acid sequence set forth in amino acids 51-56 of SEQ ID NO: 3,
and an FG loop having the amino acid sequence set forth in amino
acids 76-83 of SEQ ID NO: 3. In some embodiments, the EGFR binding
.sup.10Fn3 has an amino acid sequence at least 80, 90, 95, 98, or
100% identical to SEQ ID NO: 185, 186, 187 or 320. In some
embodiments, the IGFIR binding .sup.10Fn3 has an amino acid
sequence at least 80, 90, 95, 98, or 100% identical to SEQ ID NO:
3. In some embodiments, the E/I binder comprises an amino acid
sequence at least 80, 85, 90, 95, 98, or 100% identical to SEQ ID
NOs: 192-197.
In some embodiments, an E/I binder is an antibody-like protein
dimer comprising a) an EGFR binding .sup.10Fn3 comprising a BC loop
having the amino acid sequence set forth in amino acids 13-22 of
SEQ ID NO: 199, a DE loop having the amino acid sequence set forth
in amino acids 43-48 of SEQ ID NO: 199, and an FG loop having the
amino acid sequence set forth in amino acids 68-84 of SEQ ID NO:
199; covalently or non-covalently linked to b) an IGFIR binding
.sup.10Fn3 comprising a BC loop having the amino acid sequence set
forth in amino acids 21-30 of SEQ ID NO: 3, a DE loop having the
amino acid sequence set forth in amino acids 51-56 of SEQ ID NO: 3,
and an FG loop having the amino acid sequence set forth in amino
acids 76-83 of SEQ ID NO: 3. In some embodiments, the EGFR binding
.sup.10Fn3 has an amino acid sequence at least 80, 90, 95, 98, or
100% identical to SEQ ID NO: 198, 199, 200 or 327. In some
embodiments, the IGFIR binding .sup.10Fn3 has an amino acid
sequence at least 80, 90, 95, 98, or 100% identical to SEQ ID NO:
3. In some embodiments, the E/I binder comprises an amino acid
sequence at least 80, 85, 90, 95, 98, or 100% identical to SEQ ID
NOs: 205-210.
In some embodiments, an E/I binder is an antibody-like protein
dimer comprising an IGFIR binding .sup.10Fn3 comprising a BC loop
having the amino acid sequence X.sub.aSARLKVAX.sub.b (SEQ ID NO:
46), a DE loop having the amino acid sequence X.sub.cKNVYX.sub.d
(SEQ ID NO: 48), and an FG loop having the amino acid sequence
X.sub.eRFRDYQX.sub.f (SEQ ID NO: 50); covalently or non-covalently
linked to an EGFR binding .sup.10Fn3 comprising a BC loop having
the amino acid sequence X.sub.gDSGRGSYQX.sub.h (SEQ ID NO: 40), a
DE loop having the amino acid sequence X.sub.iGPVHX.sub.j (SEQ ID
NO: 42), and an FG loop having the amino acid sequence
X.sub.kDHKPHADGPHTYHEX.sub.l (SEQ ID NO: 44); wherein X is any
amino acid and a, b, c, d, e, f, g, h, i, j, k, and l are integers
independently selected from 0 to 5. In some embodiments, a, g, and
l are 2; b-f and i-k are 1; and h is zero. In some embodiments, a-l
are zero.
In some embodiments, an E/I binder is an antibody-like protein
dimer comprising an IGFIR binding .sup.10Fn3 comprising a BC loop
having the amino acid sequence SWSARLKVAR (SEQ ID NO: 45), a DE
loop having the amino acid sequence PKNVYT (SEQ ID NO: 47), and an
FG loop having the amino acid sequence TRFRDYQP (SEQ ID NO: 49);
covalently or non-covalently linked to an EGFR binding .sup.10Fn3
comprising a BC loop having the amino acid sequence SWDSGRGSYQ (SEQ
ID NO: 39), a DE loop having the amino acid sequence PGPVHT (SEQ ID
NO: 41), and an FG loop having the amino acid sequence
TDHKPHADGPHTYHESP (SEQ ID NO: 43).
In some embodiments, an E/I binder is an antibody-like protein
dimer comprising an IGFIR binding .sup.10Fn3 comprising a BC loop
having the amino acid sequence X.sub.aSARLKVAX.sub.b (SEQ ID NO:
46), a DE loop having the amino acid sequence X.sub.cKNVYX.sub.d
(SEQ ID NO: 48), and an FG loop having the amino acid sequence
X.sub.eRFRDYQX.sub.f (SEQ ID NO: 50); covalently or non-covalently
linked to an EGFR binding .sup.10Fn3 comprising a BC loop having
the amino acid sequence X.sub.mVAGAEDYQX.sub.n (SEQ ID NO: 34), a
DE loop having the amino acid sequence X.sub.oHDLVX.sub.p (SEQ ID
NO: 36), and an FG loop having the amino acid sequence
X.sub.qDMMHVEYTEHX.sub.r (SEQ ID NO: 38); wherein X is any amino
acid and a, b, c, d, e, f, m, n, o, p, q, and r are integers from 0
to 5, independently. In some embodiments, a and m are 2; b-f and
o-r are 1; and n is zero. In some embodiments, a-f and m-r are
zero.
In some embodiments, an E/I binder is an antibody-like protein
dimer comprising an IGFIR binding .sup.10Fn3 comprising a BC loop
having the amino acid sequence SWSARLKVAR (SEQ ID NO: 45), a DE
loop having the amino acid sequence PKNVYT (SEQ ID NO: 47), and an
FG loop having the amino acid sequence TRFRDYQP (SEQ ID NO: 49);
covalently or non-covalently linked to an EGFR binding .sup.10Fn3
comprising a BC loop having the amino acid sequence SWVAGAEDYQ (SEQ
ID NO: 33), a DE loop having the amino acid sequence PHDLVT (SEQ ID
NO: 35), and an FG loop having the amino acid sequence TDMMHVEYTEHP
(SEQ ID NO: 37).
In some embodiments, an E/I binder is an antibody-like protein
dimer comprising an IGFIR binding .sup.10Fn3 comprising a BC loop
having the amino acid sequence X.sub.aSARLKVAX.sub.b (SEQ ID NO:
46), a DE loop having the amino acid sequence X.sub.cKNVYX.sub.d
(SEQ ID NO: 48), and an FG loop having the amino acid sequence
X.sub.eRFRDYQX.sub.f (SEQ ID NO: 50); covalently or non-covalently
linked to an EGFR binding .sup.10Fn3 comprising a BC loop having
the amino acid sequence X.sub.sLPGKLRYQX.sub.t (SEQ ID NO: 60), a
DE loop having the amino acid sequence X.sub.uHDLRX.sub.w (SEQ ID
NO: 62), and an FG loop having the amino acid sequence
X.sub.yNMMHVEYSEYX.sub.z (SEQ ID NO: 64); wherein X is any amino
acid and a, b, c, d, e, f, s, t, u, w, y, and z are integers from 0
to 5, independently. In some embodiments, a and s are 2; b-f, u, w,
y and z are 1; and t is zero. In some embodiments, a-f, s-u, w, y
and z are zero.
In some embodiments, an E/I binder is an antibody-like protein
dimer comprising an IGFIR binding .sup.10Fn3 comprising a BC loop
having the amino acid sequence SWSARLKVAR (SEQ ID NO: 45), a DE
loop having the amino acid sequence PKNVYT (SEQ ID NO: 47), and an
FG loop having the amino acid sequence TRFRDYQP (SEQ ID NO: 49);
covalently or non-covalently linked to an EGFR binding .sup.10Fn3
comprising a BC loop having the amino acid sequence SWLPGKLRYQ (SEQ
ID NO: 59), a DE loop having the amino acid sequence PHDLRT (SEQ ID
NO: 61), and an FG loop having the amino acid sequence TNMMHVEYSEYP
(SEQ ID NO: 63).
In some embodiments, an E/I binder is an antibody-like protein
dimer comprising an IGFIR binding .sup.10Fn3 comprising a BC loop
having the amino acid sequence X.sub.aSARLKVAX.sub.b (SEQ ID NO:
46), a DE loop having the amino acid sequence X.sub.cKNVYX.sub.d
(SEQ ID NO: 48), and an FG loop having the amino acid sequence
X.sub.eRFRDYQX.sub.f (SEQ ID NO: 50); covalently or non-covalently
linked to an EGFR binding .sup.10Fn3 comprising a BC loop having
the amino acid sequence X.sub.gHERDGSRQX.sub.h (SEQ ID NO: 134), a
DE loop having the amino acid sequence X.sub.iGGVRX.sub.j (SEQ ID
NO: 135), and an FG loop having the amino acid sequence
X.sub.kDYFNPTTHEYIYQTTX.sub.l (SEQ ID NO: 136); wherein X is any
amino acid and a, b, c, d, e, f, g, h, i, j, k and l are integers
from 0 to 5, independently. In some embodiments, a and g are 2; b-f
and i-l are 1; and h is zero. In some embodiments, a-l are
zero.
In some embodiments, an E/I binder is an antibody-like protein
dimer comprising an IGFIR binding .sup.10Fn3 comprising a BC loop
having the amino acid sequence SWSARLKVAR (SEQ ID NO: 45), a DE
loop having the amino acid sequence PKNVYT (SEQ ID NO: 47), and an
FG loop having the amino acid sequence TRFRDYQP (SEQ ID NO: 49);
covalently or non-covalently linked to an EGFR binding .sup.10Fn3
comprising a BC loop having the amino acid sequence SWHERDGSRQ (SEQ
ID NO: 109), a DE loop having the amino acid sequence PGGVRT (SEQ
ID NO: 110), and an FG loop having the amino acid sequence
TDYFNPTTHEYIYQTTP (SEQ ID NO: 111).
In some embodiments, an E/I binder is an antibody-like protein
dimer comprising an IGFIR binding .sup.10Fn3 comprising a BC loop
having the amino acid sequence X.sub.aSARLKVAX.sub.b (SEQ ID NO:
46), a DE loop having the amino acid sequence X.sub.cKNVYX.sub.d
(SEQ ID NO: 48), and an FG loop having the amino acid sequence
X.sub.eRFRDYQX.sub.f (SEQ ID NO: 50); covalently or non-covalently
linked to an EGFR binding .sup.10Fn3 comprising a BC loop having
the amino acid sequence X.sub.gWAPVDRYQX.sub.h (SEQ ID NO: 137), a
DE loop having the amino acid sequence X.sub.iRDVYX.sub.j (SEQ ID
NO: 138), and an FG loop having the amino acid sequence
X.sub.kDYKPHADGPHTYHESX.sub.l (SEQ ID NO: 139); wherein X is any
amino acid and a, b, c, d, e, f, g, h, i, j, k and l are integers
from 0 to 5, independently. In some embodiments, a and g are 2; b-f
and i-l are 1; and h is zero. In some embodiments, a-l are
zero.
In some embodiments, an E/I binder is an antibody-like protein
dimer comprising an IGFIR binding .sup.10Fn3 comprising a BC loop
having the amino acid sequence SWSARLKVAR (SEQ ID NO: 45), a DE
loop having the amino acid sequence PKNVYT (SEQ ID NO: 47), and an
FG loop having the amino acid sequence TRFRDYQP (SEQ ID NO: 49);
covalently or non-covalently linked to an EGFR binding .sup.10Fn3
comprising a BC loop having the amino acid sequence SWWAPVDRYQ (SEQ
ID NO: 115), a DE loop having the amino acid sequence PRDVYT (SEQ
ID NO: 116), and an FG loop having the amino acid sequence
TDYKPHADGPHTYHESP (SEQ ID NO: 117).
In some embodiments, an E/I binder is an antibody-like protein
dimer comprising an IGFIR binding .sup.10Fn3 comprising a BC loop
having the amino acid sequence X.sub.aSARLKVAX.sub.b (SEQ ID NO:
46), a DE loop having the amino acid sequence X.sub.cKNVYX.sub.d
(SEQ ID NO: 48), and an FG loop having the amino acid sequence
X.sub.eRFRDYQX.sub.f (SEQ ID NO: 50); covalently or non-covalently
linked to an EGFR binding .sup.10Fn3 comprising a BC loop having
the amino acid sequence X.sub.gTQGSTHYQX.sub.h (SEQ ID NO: 146), a
DE loop having the amino acid sequence X.sub.iGMVYX.sub.j (SEQ ID
NO: 147), and an FG loop having the amino acid sequence
X.sub.kDYFDRSTHEYKYRTTX.sub.l (SEQ ID NO: 148); wherein X is any
amino acid and a, b, c, d, e, f, g, h, i, j, k and l are integers
from 0 to 5, independently. In some embodiments, a and g are 2; b-f
and i-l are 1; and h is zero. In some embodiments, a-l are
zero.
In some embodiments, an E/I binder is an antibody-like protein
dimer comprising an IGFIR binding .sup.10Fn3 comprising a BC loop
having the amino acid sequence SWSARLKVAR (SEQ ID NO: 45), a DE
loop having the amino acid sequence PKNVYT (SEQ ID NO: 47), and an
FG loop having the amino acid sequence TRFRDYQP (SEQ ID NO: 49);
covalently or non-covalently linked to an EGFR binding .sup.10Fn3
comprising a BC loop having the amino acid sequence SWTQGSTHYQ (SEQ
ID NO: 143), a DE loop having the amino acid sequence PGMVYT (SEQ
ID NO: 144), and an FG loop having the amino acid sequence
TDYFDRSTHEYKYRTTP (SEQ ID NO: 145).
In some embodiments, an E/I binder is an antibody-like protein
dimer comprising an IGFIR binding .sup.10Fn3 comprising a BC loop
having the amino acid sequence X.sub.aSARLKVAX.sub.b (SEQ ID NO:
46), a DE loop having the amino acid sequence X.sub.cKNVYX.sub.d
(SEQ ID NO: 48), and an FG loop having the amino acid sequence
X.sub.eRFRDYQX.sub.f (SEQ ID NO: 50); covalently or non-covalently
linked to an EGFR binding .sup.10Fn3 comprising a BC loop having
the amino acid sequence X.sub.gYWEGLPYQX.sub.h (SEQ ID NO: 161), a
DE loop having the amino acid sequence X.sub.iRDVNX.sub.j (SEQ ID
NO: 162), and an FG loop having the amino acid sequence
X.sub.kDWYNPDTHEYIYHTIX.sub.l (SEQ ID NO: 163); wherein X is any
amino acid and a, b, c, d, e, f, g, h, i, j, k and l are integers
from 0 to 5, independently. In some embodiments, a and g are 2; b-f
and i-l are 1; and h is zero. In some embodiments, a-l are
zero.
In some embodiments, an E/I binder is an antibody-like protein
dimer comprising an IGFIR binding .sup.10Fn3 comprising a BC loop
having the amino acid sequence SWSARLKVAR (SEQ ID NO: 45), a DE
loop having the amino acid sequence PKNVYT (SEQ ID NO: 47), and an
FG loop having the amino acid sequence TRFRDYQP (SEQ ID NO: 49);
covalently or non-covalently linked to an EGFR binding .sup.10Fn3
comprising a BC loop having the amino acid sequence SWYWEGLPYQ (SEQ
ID NO: 158), a DE loop having the amino acid sequence PRDVNT (SEQ
ID NO: 159), and an FG loop having the amino acid sequence
TDWYNPDTHEYIYHTIP (SEQ ID NO: 160).
In some embodiments, an E/I binder is an antibody-like protein
dimer comprising an IGFIR binding .sup.10Fn3 comprising a BC loop
having the amino acid sequence X.sub.aSARLKVAX.sub.b (SEQ ID NO:
46), a DE loop having the amino acid sequence X.sub.cKNVYX.sub.d
(SEQ ID NO: 48), and an FG loop having the amino acid sequence
X.sub.eRFRDYQX.sub.f (SEQ ID NO: 50); covalently or non-covalently
linked to an EGFR binding .sup.10Fn3 comprising a BC loop having
the amino acid sequence X.sub.gASNRGTYQX.sub.h (SEQ ID NO: 176), a
DE loop having the amino acid sequence X.sub.iGGVSX.sub.j (SEQ ID
NO: 177), and an FG loop having the amino acid sequence
X.sub.kDAFNPTTHEYNYFTTX.sub.l (SEQ ID NO: 178); wherein X is any
amino acid and a, b, c, d, e, f, g, h, i, j, k and l are integers
from 0 to 5, independently. In some embodiments, a and g are 2; b-f
and i-l are 1; and h is zero. In some embodiments, a-l are
zero.
In some embodiments, an E/I binder is an antibody-like protein
dimer comprising an IGFIR binding .sup.10Fn3 comprising a BC loop
having the amino acid sequence SWSARLKVAR (SEQ ID NO: 45), a DE
loop having the amino acid sequence PKNVYT (SEQ ID NO: 47), and an
FG loop having the amino acid sequence TRFRDYQP (SEQ ID NO: 49);
covalently or non-covalently linked to an EGFR binding .sup.10Fn3
comprising a BC loop having the amino acid sequence SWASNRGTYQ (SEQ
ID NO: 173), a DE loop having the amino acid sequence PGGVST (SEQ
ID NO: 174), and an FG loop having the amino acid sequence
TDAFNPTTHEYNYFTTP (SEQ ID NO: 175).
In some embodiments, an E/I binder is an antibody-like protein
dimer comprising an IGFIR binding .sup.10Fn3 comprising a BC loop
having the amino acid sequence X.sub.aSARLKVAX.sub.b (SEQ ID NO:
46), a DE loop having the amino acid sequence X.sub.cKNVYX.sub.d
(SEQ ID NO: 48), and an FG loop having the amino acid sequence
X.sub.eRFRDYQX.sub.f (SEQ ID NO: 50); covalently or non-covalently
linked to an EGFR binding .sup.10Fn3 comprising a BC loop having
the amino acid sequence X.sub.gDAPTSRYQX.sub.h (SEQ ID NO: 190), a
DE loop having the amino acid sequence X.sub.iGGLSX.sub.j (SEQ ID
NO: 191), and an FG loop having the amino acid sequence
X.sub.kDYKPHADGPHTYHESX.sub.l (SEQ ID NO: 139); wherein X is any
amino acid and a, b, c, d, e, f, g, h, i, j, k and l are integers
from 0 to 5, independently. In some embodiments, a and g are 2; b-f
and i-l are 1; and h is zero. In some embodiments, a-l are
zero.
In some embodiments, an E/I binder is an antibody-like protein
dimer comprising an IGFIR binding .sup.10Fn3 comprising a BC loop
having the amino acid sequence SWSARLKVAR (SEQ ID NO: 45), a DE
loop having the amino acid sequence PKNVYT (SEQ ID NO: 47), and an
FG loop having the amino acid sequence TRFRDYQP (SEQ ID NO: 49);
covalently or non-covalently linked to an EGFR binding .sup.10Fn3
comprising a BC loop having the amino acid sequence SWDAPTSRYQ (SEQ
ID NO: 188), a DE loop having the amino acid sequence PGGLST (SEQ
ID NO: 189), and an FG loop having the amino acid sequence
TDYKPHADGPHTYHESP (SEQ ID NO: 117).
In some embodiments, an E/I binder is an antibody-like protein
dimer comprising an IGFIR binding .sup.10Fn3 comprising a BC loop
having the amino acid sequence X.sub.aSARLKVAX.sub.b (SEQ ID NO:
46), a DE loop having the amino acid sequence X.sub.cKNVYX.sub.d
(SEQ ID NO: 48), and an FG loop having the amino acid sequence
X.sub.eRFRDYQX.sub.f (SEQ ID NO: 50); covalently or non-covalently
linked to an EGFR binding .sup.10Fn3 comprising a BC loop having
the amino acid sequence X.sub.gDAGAVTYQX.sub.h (SEQ ID NO: 203), a
DE loop having the amino acid sequence X.sub.iGGVRX.sub.j (SEQ ID
NO: 135), and an FG loop having the amino acid sequence
X.sub.kDYKPHADGPHTYHEYX.sub.l (SEQ ID NO: 204); wherein X is any
amino acid and a, b, c, d, e, f, g, h, i, j, k and l are integers
from 0 to 5, independently. In some embodiments, a and g are 2; b-f
and i-l are 1; and h is zero. In some embodiments, a-l are
zero.
In some embodiments, an E/I binder is an antibody-like protein
dimer comprising an IGFIR binding .sup.10Fn3 comprising a BC loop
having the amino acid sequence SWSARLKVAR (SEQ ID NO: 45), a DE
loop having the amino acid sequence PKNVYT (SEQ ID NO: 47), and an
FG loop having the amino acid sequence TRFRDYQP (SEQ ID NO: 49);
covalently or non-covalently linked to an EGFR binding .sup.10Fn3
comprising a BC loop having the amino acid sequence SWDAGAVTYQ (SEQ
ID NO: 201), a DE loop having the amino acid sequence PGGVRT (SEQ
ID NO: 110), and an FG loop having the amino acid sequence
TDYKPHADGPHTYHEYP (SEQ ID NO: 202).
In some embodiments, an E/I binder is an antibody-like protein
dimer comprising a) an IGFIR binding .sup.10Fn3 comprising a BC
loop having the amino acid sequence set forth in amino acids 23-29
of SEQ ID NO: 3, a DE loop having the amino acid sequence set forth
in amino acids 52-55 of SEQ ID NO: 3, and an FG loop having the
amino acid sequence set forth in amino acids 77-82 of SEQ ID NO: 3;
covalently or non-covalently linked to b) an EGFR binding
.sup.10Fn3 comprising a BC, DE and FG loop as set forth in any one
of SEQ ID NOs: 219-327 (see e.g., FIG. 45 wherein the BC, DE and FG
loop sequences for each EGFR binding .sup.10Fn3 are underlined). In
some embodiments, an E/I binder is an antibody-like protein dimer
comprising a) an IGFIR binding .sup.10Fn3 comprising a BC loop
having the amino acid sequence set forth in amino acids 23-29 of
SEQ ID NO: 3, a DE loop having the amino acid sequence set forth in
amino acids 52-55 of SEQ ID NO: 3, and an FG loop having the amino
acid sequence set forth in amino acids 77-82 of SEQ ID NO: 3;
covalently or non-covalently linked to b) an EGFR binding
.sup.10Fn3 comprising a BC loop having the amino acid sequence set
forth in amino acids corresponding to amino acid residues 23-30 of
SEQ ID NO: 1 of any one of SEQ ID NOs: 5-8, 52, 66-68, 106-108,
112-114, 140-142, 155-157, 170-172, 182, 185-187, 198-200, or
219-327, a DE loop having the amino acid sequence set forth in
amino acids corresponding to amino acid residues 52-55 of SEQ ID
NO: 1 of any one of SEQ ID NOs: 5-8, 52, 66-68, 106-108, 112-114,
140-142, 155-157, 170-172, 182, 185-187, 198-200, or 219-327, and
an FG loop having the amino acid sequence set forth in amino acids
corresponding to amino acid residues 77-86 of SEQ ID NO: 15-8, 52,
66-68, 106-108, 112-114, 140-142, 155-157, 170-172, 182, 185-187,
198-200, or 219-327. In some embodiments, the EGFR binding
.sup.10Fn3 of the antibody-like protein dimer comprises an amino
acid sequence at least 80, 90, 95, or 100% identical to the amino
acid sequence of amino acid residues corresponding to E9 of SEQ ID
NO: 1 through T94 of SEQ ID NO: 1 of any one of SEQ ID NOs: 5-8,
52, 66-68, 106-108, 112-114, 140-142, 155-157, 170-172, 182,
185-187, 198-200, or 219-327. In some embodiments, the IGFIR
binding .sup.10Fn3 of the antibody-like protein dimer has an amino
acid sequence at least 80, 90, 95, 98, 99, or 100% identical to the
amino acid sequence of amino acid residues corresponding to E9 of
SEQ ID NO: 1 through T94 of SEQ ID NO: 1 of SEQ ID NO: 3. In some
embodiments, the E/I binder comprises an amino acid sequence at
least 80, 85, 90, 95, 98, 99, or 100% identical to any one of SEQ
ID NOs: 20-31, 53-58, 87-92, 98-105, 118-133, 149-154, 164-169,
179-184, 192-197, 205-210 and 211-216.
Preferably, X as defined herein is a naturally occurring amino
acid.
In certain embodiments, the E binders, or the E and/or I monomers
of the E/I binders described herein may contain a Ser to Cys amino
acid substitution at a position corresponding to serine 62 or
serine 91 of SEQ ID NO: 1.
In certain aspects, the disclosure provides short peptide sequences
that mediate EGFR binding. Examples of such sequences include the
amino acid residues that correspond to the BC, DE, and FG loops
from SEQ ID NOs: 5, 7, 82, 106, 112, 141, 156, 171, 186 and 199.
Other examples of such sequences include the amino acid residues
that correspond to the BC, DE, and FG loops from SEQ ID NOs:
219-327. In some embodiments, the peptides bind to their respective
ligand with a dissociation constant (K.sub.D) of less than 500 nM,
100 nM, 50 nM, 5 nM or less. Such sequences may mediate ligand
binding in an isolated form or when inserted into a particular
protein structure, such as an immunoglobulin or immunoglobulin-like
domain.
In one embodiment, an antibody-like protein dimer comprises a
polypeptide having the structure A-B-C, wherein A is a polypeptide
comprising, consisting essentially of, or consisting of a
.sup.10Fn3 domain that binds to EGFR, B is a polypeptide linker,
and C is a polypeptide comprising, consisting essentially of, or
consisting of a .sup.10Fn3 domain that binds to IGF-IR. In another
embodiment, a antibody-like protein dimer comprises a polypeptide
having the structure A-B-C, wherein A is a polypeptide comprising,
consisting essentially of, or consisting of a .sup.10Fn3 domain
that binds to IGF-IR, B is a polypeptide linker, and C is a
polypeptide comprising, consisting essentially of, or consisting of
a .sup.10Fn3 domain that binds to EGFR. Specific examples of
antibody-like protein dimers having the structure A-B-C are
polypeptides comprising (i) a polypeptide having an amino acid
sequence set forth in any one of SEQ ID NOs: 20-31, 53-58, 87-92,
98-105, 118-133, 149-154, 164-169, 179-184, 192-197, 205-210 and
211-216, or (ii) a polypeptide comprising an amino acid sequence at
least 85%, 90%, 95%, 97%, 98%, or 99% identical to any one of the
amino acid sequences set forth in SEQ ID NOs: 20-31, 53-58, 87-92,
98-105, 118-133, 149-154, 164-169, 179-184, 192-197, 205-210 and
211-216.
In certain embodiments, the A or C region is a polypeptide
comprising a .sup.10Fn3 domain that binds to EGFR; wherein the
.sup.10Fn3 domain has the structure from N-terminus to C-terminus:
beta strand A, loop AB, beta strand B, loop BC, beta strand C, loop
CD, beta strand D, loop DE, beta strand E, loop EF, beta strand F,
loop FG, beta strand G; wherein: (i) the BC loop has the amino acid
sequence of SEQ ID NO: 33 or 34, the DE loop has the amino acid
sequence of SEQ ID NO: 35 or 36, and the FG loop has the amino acid
sequence of SEQ ID NO: 37 or 38, (ii) the BC loop has the amino
acid sequence of SEQ ID NO: 39 or 40, the DE loop has the amino
acid sequence of SEQ ID NO: 41 or 42, and the FG loop has the amino
acid sequence of SEQ ID NO: 43 or 44, (iii) the BC loop has the
amino acid sequence of SEQ ID NO: 59 or 60, the DE loop has the
amino acid sequence of SEQ ID NO: 61 or 62, and the FG loop has the
amino acid sequence of SEQ ID NO: 63 or 64, (iv) the BC loop has
the amino acid sequence of SEQ ID NO: 109 or 134, the DE loop has
the amino acid sequence of SEQ ID NO: 110 or 135, and the FG loop
has the amino acid sequence of SEQ ID NO: 111 or 136, (v) the BC
loop has the amino acid sequence of SEQ ID NO: 115 or 137, the DE
loop has the amino acid sequence of SEQ ID NO: 116 or 138, and the
FG loop has the amino acid sequence of SEQ ID NO: 117 or 139, (vi)
the BC loop has the amino acid sequence of SEQ ID NO: 143 or 146,
the DE loop has the amino acid sequence of SEQ ID NO: 144 or 147,
and the FG loop has the amino acid sequence of SEQ ID NO: 145 or
148, (vii) the BC loop has the amino acid sequence of SEQ ID NO:
158 or 161, the DE loop has the amino acid sequence of SEQ ID NO:
159 or 162, and the FG loop has the amino acid sequence of SEQ ID
NO: 160 or 163, (viii) the BC loop has the amino acid sequence of
SEQ ID NO: 173 or 176, the DE loop has the amino acid sequence of
SEQ ID NO: 174 or 177, and the FG loop has the amino acid sequence
of SEQ ID NO: 175 or 178, (ix) the BC loop has the amino acid
sequence of SEQ ID NO: 188 or 190, the DE loop has the amino acid
sequence of SEQ ID NO: 189 or 191, and the FG loop has the amino
acid sequence of SEQ ID NO: 117 or 139, (x) the BC loop has the
amino acid sequence of SEQ ID NO: 201 or 203, the DE loop has the
amino acid sequence of SEQ ID NO: 110 or 135, and the FG loop has
the amino acid sequence of SEQ ID NO: 202 or 204, or (xi) the BC,
DE and FG loops have the amino acid sequences as set forth in any
one of SEQ ID NOs: 219-327 (see e.g., FIG. 45 wherein the BC, DE
and FG loops for each of SEQ ID NOs: 219-327 are underlined);
wherein the .sup.10Fn3 domain folds into an antibody heavy chain
variable region-like structure; and wherein the polypeptide binds
to EGFR with a K.sub.D of less than 100 nM. The .sup.10Fn3 domain
that binds to EGFR preferably folds into a structure wherein the 7
beta strands are distributed between two beta sheets that pack
against each other forming a stable core and wherein the beta
strands are connected by the six loops which are solvent exposed.
In exemplary embodiments, the .sup.10Fn3 domain is from 80-150
amino acids in length.
In exemplary embodiments, the A or C region is a .sup.10Fn3 domain
that binds to EGFR with a K.sub.D of less than 100 nM having a
sequence selected from the group consisting of SEQ ID NO: 83-85 and
466-472 as set forth below:
TABLE-US-00007 (SEQ ID NO: 83)
EVVAATX.sub.n1SLLIX.sub.a1SWVAGAEDYQX.sub.a2YYRITYGEX.sub.n2QEFTVX.sub.a3
PHDLVTX.sub.a4ATIX.sub.n3DYTITVYAVX.sub.a5TDMMHVEYTEHPX.sub.a6ISINYRT;
(SEQ ID NO: 84)
EVVAATX.sub.n1SLLIX.sub.a1SWDSGRGSYQX.sub.a2YYRITYGEX.sub.n2QEFTVX.sub.a3
PGPVHTX.sub.a4ATIX.sub.n3DYTITVYAVX.sub.a5TDHKPHADGPHTYHESPX.sub.a6
ISINYRT; or (SEQ ID NO: 85)
EVVAATX.sub.n1SLLIX.sub.a1SWLPGKLRYQX.sub.a2YYRITYGEX.sub.n2QEFTVX.sub.a3
PHDLRTX.sub.a4ATIX.sub.n3DYTITVYAVX.sub.a5TNMMHVEYSEYPX.sub.a6ISINYRT.
(SEQ ID NO: 466)
EVVAATX.sub.n1SLLIX.sub.a1SWHERDGSRQX.sub.a2YYRITYGEX.sub.n2QEFTVX.sub.a3
PGGVRTX.sub.a4ATIX.sub.n3DYTITVYAVX.sub.a5TDYFNPTTHEYIYQTTPX.sub.a6
ISINYRT. (SEQ ID NO: 467)
EVVAATX.sub.n1SLLIX.sub.a1SWWAPVDRYQX.sub.a2YYRITYGEX.sub.n2QEFTVX.sub.a3
PRDVYTX.sub.a4ATIX.sub.n3DYTITVYAVX.sub.a5TDYKPHADGPHTYHESPX.sub.a6
ISINYRT. (SEQ ID NO: 468)
EVVAATX.sub.n1SLLIX.sub.a1SWTQGSTHYQX.sub.a2YYRITYGEX.sub.n2QEFTVX.sub.a3
PGMVYTX.sub.a4ATIX.sub.n3DYTITVYAVX.sub.a5TDYFDRSTHEYKYRTTPX.sub.a6
ISINYRT. (SEQ ID NO: 469)
EVVAATX.sub.n1SLLIX.sub.a1SWYWEGLPYQX.sub.a2YYRITYGEX.sub.n2QEFTVX.sub.a3
PRDVNTX.sub.a4ATIX.sub.n3DYTITVYAVX.sub.a5TDWYNPDTHEYIYHTIPX.sub.a6
ISINYRT. (SEQ ID NO: 470)
EVVAATX.sub.n1SLLIX.sub.a1SWASNRGTYQX.sub.a2YYRITYGEX.sub.n2QEFTVX.sub.a3
PGGVSTX.sub.a4ATIX.sub.n3DYTITVYAVX.sub.a5TDAFNPTTHEYNYFTTPX.sub.a6
ISINYRT. (SEQ ID NO: 471)
EVVAATX.sub.n1SLLIX.sub.a1SWDAPTSRYQX.sub.a2YYRITYGEX.sub.n2QEFTVX.sub.a3
PGGLSTX.sub.a4ATIX.sub.n3DYTITVYAVX.sub.a5TDYKPHADGPHTYHESPX.sub.a6
ISINYRT. (SEQ ID NO: 472)
EVVAATX.sub.n1SLLIX.sub.a1SWDAGAVTYQX.sub.a2YYRITYGEX.sub.n2QEFTVX.sub.a3
PGGVRTX.sub.a4ATIX.sub.n3DYTITVYAVX.sub.a5TDYKPHADGPHTYHEYPX.sub.a6
ISINYRT.
In SEQ ID NOs: 83-85 and 466-472, the BC, DE and FG loops have a
fixed sequence as shown in bold, or a sequence at least 75%, 80%,
85%, 90%, 95%, 97%, 98%, or 99% identical to the sequences shown in
bold, the AB loop is represented by X.sub.n1, the CD is represented
by X.sub.n2, and EF loop is represented by X.sub.n3, and the beta
strands A-G are underlined. X represents any amino acid and the
subscript following the X represents an integer of the number of
amino acids. In particular, n1 may be anywhere from 1-15, 2-15,
1-10, 2-10, 1-8, 2-8, 1-5, 2-5, 1-4, 2-4, 1-3, 2-3, or 1-2 amino
acids; n2 and n3 may each independently be anywhere from 2-20,
2-15, 2-10, 2-8, 5-20, 5-15, 5-10, 5-8, 6-20, 6-15, 6-10, 6-8, 2-7,
5-7, or 6-7 amino acids; and a1-a6 may each independently comprise
from 0-10, 0-5, 1-10, 1-5, or 2-5 amino acids. In preferred
embodiments, n1 is 2 amino acids, n2 is 7 amino acids, n3 is 7
amino acids, and a1-a6 is 0 amino acids. The sequences of the beta
strands may have anywhere from 0 to 10, from 0 to 8, from 0 to 6,
from 0 to 5, from 0 to 4, from 0 to 3, from 0 to 2, or from 0 to 1
substitutions, deletions or additions across all 7 scaffold regions
relative to the corresponding amino acids shown in SEQ ID NO: 1. In
an exemplary embodiment, the sequences of the beta strands may have
anywhere from 0 to 10, from 0 to 8, from 0 to 6, from 0 to 5, from
0 to 4, from 0 to 3, from 0 to 2, or from 0 to 1 conservative
substitutions across all 7 scaffold regions relative to the
corresponding amino acids shown in SEQ ID NO: 1. In certain
embodiments, the core amino acid residues are fixed and any
substitutions, conservative substitutions, deletions or additions
occur at residues other than the core amino acid residues. In
certain embodiments, the EGFR binder is represented by one of the
following amino acid sequences:
TABLE-US-00008 (SEQ ID NO: 66)
EVVAATPTSLLISWVAGAEDYQYYRITYGETGGNSPVQEFTVPHDLV
TATISGLKPGVDYTITVYAVTDMMHVEYTEHPISINYRT; (SEQ ID NO: 67)
EVVAATPTSLLISWDSGRGSYQYYRITYGETGGNSPVQEFTVPGPVH
TATISGLKPGVDYTITVYAVTDHKPHADGPHTYHESPISINYRT; (SEQ ID NO: 68)
EVVAATPTSLLISWLPGKLRYQYYRITYGETGGNSPVQEFTVPHDLR
TATISGLKPGVDYTITVYAVTNMMHVEYSEYPISINYRT; (SEQ ID NO: 108)
EVVAATPTSLLISWHERDGSRQYYRITYGETGGNSPVQEFTVPGGVR
TATISGLKPGVDYTITVYAVTDYFNPTTHEYIYQTTPISINYRT; or (SEQ ID NO: 114)
EVVAATPTSLLISWWAPVDRYQYYRITYGETGGNSPVQEFTVPRDVY
TATISGLKPGVDYTITVYAVTDYKPHADGPHTYHESPISINYRT. (SEQ ID NO: 141)
EVVAATPTSLLISWTQGSTHYQYYRITYGETGGNSPVQEFTVPGMVY
TATISGLKPGVDYTITVYAVTDYFDRSTHEYKYRTTPISINYRT (SEQ ID NO: 156)
EVVAATPTSLLISWYWEGLPYQYYRITYGETGGNSPVQEFTVPRDVN
TATISGLKPGVDYTITVYAVTDWYNPDTHEYIYHTIPISINYRT (SEQ ID NO: 171)
EVVAATPTSLLISWASNRGTYQYYRITYGETGGNSPVQEFTVPGGVS
TATISGLKPGVDYTITVYAVTDAFNPTTHEYNYFTTPISINYRT (SEQ ID NO: 186)
EVVAATPTSLLISWDAPTSRYQYYRITYGETGGNSPVQEFTVPGGLS
TATISGLKPGVDYTITVYAVTDYKPHADGPHTYHESPISINYRT E112 (SEQ ID NO: 199)
EVVAATPTSLLISWDAGAVTYQYYRITYGETGGNSPVQEFTVPGGVR
TATISGLKPGVDYTITVYAVTDYKPHADGPHTYHEYPISINYRT
In SEQ ID NOs: 66-68, 108, 114, 141, 156, 171, 186 and 199, the
sequence of the BC, DE and FG loops have a fixed sequence as shown
in bold, or a sequence at least 75%, 80%, 85%, 90%, 95%, 97%, 98%,
or 99% identical to the sequences shown in bold, and the remaining
sequence which is underlined (e.g., the sequence of the 7 beta
strands and the AB, CD and EF loops) has anywhere from 0 to 20,
from 0 to 15, from 0 to 10, from 0 to 8, from 0 to 6, from 0 to 5,
from 0 to 4, from 0 to 3, from 0 to 2, or from 0 to 1
substitutions, conservative substitutions, deletions or additions
relative to the corresponding amino acids shown in SEQ ID NO:
66-68, 108, 114, 141, 156, 171, 186 and 199. In certain
embodiments, the core amino acid residues are fixed and any
substitutions, conservative substitutions, deletions or additions
occur at residues other than the core amino acid residues. The
.sup.10Fn3 domain that binds to EGFR may optionally comprise an
N-terminal extension of from 1-20, 1-15, 1-10, 1-8, 1-5, 1-4, 1-3,
1-2, or 1 amino acids in length. Exemplary N-terminal extensions
include (represented by the single letter amino acid code) M, MG,
G, MGVSDVPRDL (SEQ ID NO: 69), GVSDVPRDL (SEQ ID NO: 70), and
VSDVPRDL (SEQ ID NO: 71), or N-terminal truncations of any one of
SEQ ID NOs: 69, 70, or 71. Other suitable N-terminal extensions
include, for example, X.sub.nSDVPRDL (SEQ ID NO: 72), X.sub.nDVPRDL
(SEQ ID NO: 73), X.sub.nVPRDL (SEQ ID NO: 74), X.sub.nPRDL (SEQ ID
NO: 75), X.sub.nRDL (SEQ ID NO: 76), X.sub.nDL (SEQ ID NO: 77), or
X.sub.nL, wherein n=0, 1 or 2 amino acids, wherein when n=1, X is
Met or Gly, and when n=2, X is Met-Gly. The .sup.10Fn3 domain that
binds to EGFR may optionally comprise a C-terminal tail. Exemplary
C-terminal tails include polypeptides that are from 1-20, 1-15,
1-10, 1-8, 1-5, 1-4, 1-3, 1-2, or 1 amino acids in length. Specific
examples of C-terminal tails include EIDKPSQ (SEQ ID NO: 9),
EIDKPCQ (SEQ ID NO: 10), and EIDK (SEQ ID NO: 78). In other
embodiments, suitable C-terminal tails may be a C-terminally
truncated fragment of SEQ ID NOs: 9, 10 or 78, including, for
example, one of the following amino acid sequences (represented by
the single letter amino acid code): E, EI, EID, EIDKP (SEQ ID NO:
79), EIDKPS (SEQ ID NO: 80), or EIDKPC (SEQ ID NO: 81). Other
suitable C-terminal tails include, for example, ES, EC, EGS, EGC,
EGSGS (SEQ ID NO: 96), EGSGC (SEQ ID NO: 97), or EIEK (SEQ ID NO:
217). In certain embodiments, the .sup.10Fn3 domain that binds to
EGFR comprises both an N-terminal extension and a C-terminal tail.
In exemplary embodiments, the A region comprises an N-terminal
extension beginning with Gly or Met-Gly and a C-terminal extension
that does not contain a cysteine residue and the B region comprises
an N-terminal extension that does not start with a Met and a
C-terminal extension that comprises a cysteine residue. Specific
examples of .sup.10Fn3 domains that bind to EGFR are polypeptides
comprising (i) a polypeptide having an amino acid sequence set
forth in any one of SEQ ID NOs: 5-8, 52, 66-68, 82-85, 106-108,
112-114, 140-142, 155-157, 170-172, 185-187, 198-200, and 219-327,
or (ii) a polypeptide comprising an amino acid sequence at least
85%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid
sequence set forth in any one of SEQ ID NOs: 5-8, 52, 66-68, 82-85,
106-108, 112-114, 140-142, 155-157, 170-172, 185-187, 198-200, and
219-327.
In certain embodiments, the A or C region is a polypeptide
comprising a .sup.10Fn3 domain that binds to IGF-IR, wherein the
.sup.10Fn3 domain has the structure from N-terminus to C-terminus;
beta strand A, loop AB, beta strand B, loop BC, beta strand C, loop
CD, beta strand D, loop DE, beta strand E, loop EF, beta strand F,
loop FG, beta strand G, wherein the BC loop has the amino acid
sequence of SEQ ID NO: 45 or 46, the DE loop has the amino acid
sequence of SEQ ID NO: 47 or 48, and the FG loop has the amino acid
sequence of SEQ ID NO: 49 or 50, wherein the .sup.10Fn3 domain
folds into an antibody heavy chain variable region-like structure,
and wherein the polypeptide binds to IGF-IR with a K.sub.D of less
than 100 nM. The .sup.10Fn3 domain that binds to IGF-IR preferably
folds into a structure wherein the 7 beta strands are distributed
between two beta sheets that pack against each other forming a
stable core and wherein the beta strands are connected by the six
loops which are solvent exposed. In exemplary embodiments, the
.sup.10Fn3 domain is from 80-150 amino acids in length.
In exemplary embodiments, the A or C region is a .sup.10Fn3 domain
that binds to IGF-IR with a K.sub.D of less than 100 nM having the
sequence set forth below:
TABLE-US-00009 (SEQ ID NO: 86)
EVVAATX.sub.n1SLLIX.sub.a1SWSARLKVARX.sub.a2YYRITYGEX.sub.n2QEFTVX.sub.a3
PKNVYTX.sub.a4ATIX.sub.n3DYTITVYAVX.sub.a5TRFRDYQPX.sub.a6ISINYRT.
In SEQ ID NO: 86, the BC, DE and FG loops have a fixed sequence as
shown in bold, or a sequence at least 75%, 80%, 85%, 90%, 95%, 97%,
98%, or 99% identical to the sequences shown in bold, the AB loop
is represented by X.sub.n1, the CD loop is represented by X.sub.n2,
and the EF loop is represented by X.sub.n3, and the beta strands
A-G are underlined. X represents any amino acid and the subscript
following the X represents an integer of the number of amino acids.
In particular, n1 may be anywhere from 1-15, 2-15, 1-10, 2-10, 1-8,
2-8, 1-5, 2-5, 1-4, 2-4, 1-3, 2-3, or 1-2 amino acids; n2 and n3
may each independently be anywhere from 2-20, 2-15, 2-10, 2-8,
5-20, 5-15, 5-10, 5-8, 6-20, 6-15, 6-10, 6-8, 2-7, 5-7, or 6-7
amino acids; and a1-a6 may each independently comprise from 0-10,
0-5, 1-10, 1-5, or 2-5 amino acids. In preferred embodiments, n1 is
2 amino acids, n2 is 7 amino acids, n3 is 7 amino acids, and a1-a6
is 0 amino acids. The sequences of the beta strands may have
anywhere from 0 to 10, from 0 to 8, from 0 to 6, from 0 to 5, from
0 to 4, from 0 to 3, from 0 to 2, or from 0 to 1 substitutions,
deletions or additions across all 7 scaffold regions relative to
the corresponding amino acids shown in SEQ ID NO: 1. In an
exemplary embodiment, the sequences of the beta strands may have
anywhere from 0 to 10, from 0 to 8, from 0 to 6, from 0 to 5, from
0 to 4, from 0 to 3, from 0 to 2, or from 0 to 1 conservative
substitutions across all 7 scaffold regions relative to the
corresponding amino acids shown in SEQ ID NO: 1. In certain
embodiments, the core amino acid residues are fixed and any
substitutions, conservative substitutions, deletions or additions
occur at residues other than the core amino acid residues. In
certain embodiments, the IGF-IR binder is represented by the
following amino acid sequence:
TABLE-US-00010 (SEQ ID NO: 65)
EVVAATPTSLLISWSARLKVARYYRITYGETGGNSPVQEFTVPKNVY
TATISGLKPGVDYTITVYAVTRFRDYQPISINYRT.
In SEQ ID NO: 65, the sequence of the BC, DE and FG loops have a
fixed sequence as shown in bold, or a sequence at least 75%, 80%,
85%, 90%, 95%, 97%, 98%, or 99% identical to the sequences shown in
bold, and the remaining sequence which is underlined (e.g., the
sequence of the 7 beta strands and the AB, CD and EF loops) has
anywhere from 0 to 20, from 0 to 15, from 0 to 10, from 0 to 8,
from 0 to 6, from 0 to 5, from 0 to 4, from 0 to 3, from 0 to 2, or
from 0 to 1 substitutions, conservative substitutions, deletions or
additions relative to the corresponding amino acids shown in SEQ ID
NO: 65. In certain embodiments, the core amino acid residues are
fixed and any substitutions, conservative substitutions, deletions
or additions occur at residues other than the core amino acid
residues. The .sup.10Fn3 domain that binds to IGF-IR may optionally
comprise an N-terminal extension of from 1-20, 1-15, 1-10, 1-8,
1-5, 1-4, 1-3, 1-2, or 1 amino acids in length. Exemplary
N-terminal extensions include (represented by the single letter
amino acid code) M, MG, G, MGVSDVPRDL (SEQ ID NO: 69), GVSDVPRDL
(SEQ ID NO: 70), and VSDVPRDL (SEQ ID NO: 71), or N-terminal
truncations of any one of SEQ ID NOs: 69, 70, or 71. Other suitable
N-terminal extensions include, for example, X.sub.nSDVPRDL (SEQ ID
NO: 72), X.sub.nDVPRDL (SEQ ID NO: 73), X.sub.nVPRDL (SEQ ID NO:
74), X.sub.nPRDL (SEQ ID NO: 75), X.sub.nRDL (SEQ ID NO: 76),
X.sub.n.quadrature.L (SEQ ID NO: 77), or X.sub.nL, wherein n=0, 1
or 2 amino acids, wherein when n=1, X is Met or Gly, and when n=2,
X is Met-Gly. The .sup.10Fn3 domain that binds to IGF-IR may
optionally comprise a C-terminal tail. Exemplary C-terminal tails
include polypeptides that are from 1-20, 1-15, 1-10, 1-8, 1-5, 1-4,
1-3, 1-2, or 1 amino acids in length. Specific examples of
C-terminal tails include EIDKPSQ (SEQ ID NO: 9), EIDKPCQ (SEQ ID
NO: 10), and EIDK (SEQ ID NO: 78). In other embodiments, suitable
C-terminal tails may be a C-terminally truncated fragment of SEQ ID
NOs: 9, 10 or 78, including, for example, one of the following
amino acid sequences (represented by the single letter amino acid
code): E, EI, EID, EIDKP (SEQ ID NO: 79), EIDKPS (SEQ ID NO: 80),
or EIDKPC (SEQ ID NO: 81). Other suitable C-terminal tails include,
for example, ES, EC, EGS, EGC, EGSGS (SEQ ID NO: 96), EGSGC (SEQ ID
NO: 97), or EIEK (SEQ ID NO: 217). In certain embodiments, the
.sup.10Fn3 domain that binds to IGF-IR comprises both an N-terminal
extension and a C-terminal tail. In exemplary embodiments, the A
region comprises an N-terminal extension beginning with Gly or
Met-Gly and a C-terminal extension that does not contain a cysteine
residue and the B region comprises an N-terminal extension that
does not start with a Met and a C-terminal extension that comprises
a cysteine residue. Specific examples of .sup.10Fn3 domains that
bind to IGF-IR are polypeptides comprising (i) a polypeptide having
an amino acid sequence set forth in any one of SEQ ID NOs: 3, 4, 65
or 86, or (ii) a polypeptide comprising an amino acid sequence at
least 85%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid
sequence set forth in any one of SEQ ID NOs: 3, 4, 65 or 86.
The B region is a linker as described further herein. In exemplary
embodiments, the B region is a polypeptide linker Exemplary
polypeptide linkers include polypeptides having from 1-20, 1-15,
1-10, 1-8, 1-5, 1-4, 1-3, or 1-2 amino acids. Specific examples of
suitable polypeptide linkers are described further herein and
include, for example, linkers having a sequence selected from the
group consisting of SEQ ID NOs: 11-19, 51, 93-95 and 218. In
certain embodiments, the linker may be a C-terminal tail
polypeptide as described herein, an N-terminal extension
polypeptide as described herein, or a combination thereof.
In one embodiment, an antibody-like protein dimer comprises a
polypeptide having the structure
X.sub.1-A-X.sub.2--B--X.sub.3--C--X.sub.4, wherein X.sub.1 is an
optional N-terminal extension, A is a .sup.10Fn3 domain that binds
to EGFR, X.sub.2 is an optional C-terminal tail, B is a polypeptide
linker, X.sub.3 is an optional N-terminal extension, C is a
.sup.10Fn3 domain that binds to IGF-IR, and X.sub.4 is an optional
C-terminal tail. In another embodiment, an antibody-like protein
dimer comprises a polypeptide having the structure
X.sub.1-A-X.sub.2--B--X.sub.3--C--X.sub.4, wherein X.sub.1 is an
optional N-terminal extension, A is a .sup.10Fn3 domain that binds
to IGF-IR, X.sub.2 is an optional C-terminal tail, B is a
polypeptide linker, X.sub.3 is an optional N-terminal extension, C
is a .sup.10Fn3 domain that binds to EGFR, and X.sub.4 is an
optional C-terminal tail. Specific examples of suitable N-terminal
extensions and C-terminal tails are described above. In certain
embodiments, one or more of X.sub.1, X.sub.2, B, X.sub.3 or X.sub.4
may comprise an amino acid residue suitable for pegylation, such as
a cysteine or lysine residue. In exemplary embodiments, X.sub.4
comprises at least one amino acid suitable for pegylation, such as
a cysteine or lysine residue. Specific examples of suitable
polypeptide linkers are described further below. Specific examples
of antibody-like protein dimers having the structure
X.sub.1-A-X.sub.2--B--X.sub.3--C--X.sub.4 are polypeptides
comprising (i) a polypeptide having the amino acid sequence set
forth in any one of SEQ ID NOs: 20-31, 53-58, 87-92, 98-105,
118-133, 149-154, 164-169, 179-184, 192-197, 205-210 and 211-216,
or (ii) a polypeptide comprising an amino acid sequence at least
85%, 90%, 95%, 97%, 98%, or 99% identical to the amino acid
sequence set forth in any one of SEQ ID NOs: 20-31, 53-58, 87-92,
98-105, 118-133, 149-154, 164-169, 179-184, 192-197, 205-210 and
211-216.
In certain embodiments, it may be desirable to tune the potency of
one .sup.10Fn3 binding domain relative to the other .sup.10Fn3
binding domain in the antibody-like protein dimers described
herein. For example, if the binding affinity of the first
.sup.10Fn3 domain is significantly higher than the binding affinity
of the second .sup.10Fn3 domain, the biological effect of the first
.sup.10Fn3 domain could overwhelm the effects of the second of
second .sup.10Fn3 domain. Accordingly, in certain embodiments, it
may be desirable for the binding affinities of the first and second
.sup.10Fn3 domains of an antibody-like protein dimer to be similar
to each other, e.g., binding affinities within 100-fold, 30-fold,
10-fold, 3-fold, 1-fold, 0.3-fold or 0.1-fold, of each other, or
binding affinities within 0.1-fold to 10-fold, within 0.3-fold to
10-fold, within 0.1-fold to 3-fold, within 0.3-fold to 3-fold,
within 0.1-fold to 1-fold, within 0.3-fold to 1-fold, within 1-fold
to 10-fold, within 3-fold to 10-fold, within 3-fold to 30-fold, or
within 1-fold to 3-fold of each other.
Conjugation
Multimers of antibody-like proteins may be covalently or
non-covalently linked. In some embodiments, an EGFR binding
.sup.10Fn3 may be directly or indirectly linked to an IGFIR binding
.sup.10Fn3 via a polypeptide linker Suitable linkers for joining
Fn3 are those which allow the separate domains to fold
independently of each other forming a three dimensional structure
that permits high affinity binding to a target molecule.
The disclosure provides a number of suitable linkers that meet
these requirements, including glycine-serine based linkers,
glycine-proline based linkers, as well as the linker having the
amino acid sequence PSTSTST (SEQ ID NO: 12). The Examples described
herein demonstrate that Fn3 domains joined via polypeptide linkers
retain their target binding function. In some embodiments, the
linker is a glycine-serine based linker. These linkers comprise
glycine and serine residues and may be between 8 and 50, 10 and 30,
and 10 and 20 amino acids in length. Examples include linkers
having an amino acid sequence GSGSGSGSGSGSGSGSGSGS (SEQ ID NO: 11),
GSGSGSGSGS (SEQ ID NO: 13), GGGGS GGGGS GGGGS (SEQ ID NO: 14),
GGGGS GGGGS GGGGS GGGGS (SEQ ID NO: 15), GGGGS GGGGS GGGGS GGGGS
GGGGS (SEQ ID NO: 16), or GGGGSGGGGSGGGSG (SEQ ID NO: 17). In some
embodiments, the linker is a glycine-proline based linker. These
linkers comprise glycine and proline residues and may be between 3
and 30, 10 and 30, and 3 and 20 amino acids in length. Examples
include linkers having an amino acid sequence GPGPGPG (SEQ ID NO:
18), GPGPGPGPGPG (SEQ ID NO: 19), and GPG (SEQ ID NO: 51). In some
embodiments, the linker is a proline-alanine based linker. These
linkers comprise proline and alanine residues and may be between 3
and 30, 10 and 30, 3 and 20 and 6 and 18 amino acids in length.
Examples of such linkers include SEQ ID NOs: 93, 94 and 95. It is
contemplated, that the optimal linker length and amino acid
composition may be determined by routine experimentation by methods
well known in the art.
In some embodiments, multimers of antibody-like proteins are linked
via a polypeptide linker having a protease site that is cleavable
by a protease in the blood or target tissue. Such embodiments can
be used to release two or more therapeutic proteins for better
delivery or therapeutic properties or more efficient production
compared to separately producing such proteins.
Additional linkers or spacers, e.g., SEQ ID NOs: 9 and 10, may be
introduced at the C-terminus of a Fn3 domain between the Fn3 domain
and the polypeptide linker. Additional linkers or spacers may be
introduced at the N-terminus of a Fn3 domain between the Fn3 domain
and the polypeptide linker.
In some embodiments, multimers of antibody-like proteins may be
directly or indirectly linked via a polymeric linker Polymeric
linkers can be used to optimally vary the distance between each
protein moiety to create a protein with one or more of the
following characteristics: 1) reduced or increased steric hindrance
of binding of one or more protein domain when binding to a protein
of interest, 2) increased protein stability or solubility, 3)
decreased protein aggregation, and 4) increased overall avidity or
affinity of the protein.
In some embodiments, multimers of antibody-like proteins are linked
via a biocompatible polymer such as a polymeric sugar. The
polymeric sugar can include an enzymatic cleavage site that is
cleavable by an enzyme in the blood or target tissue. Such
embodiments can be used to release two or more therapeutic proteins
for better delivery or therapeutic properties or more efficient
production compared to separately producing such proteins
In some embodiments, multimers of antibody-like proteins are linked
via a polyoxyalkylene, in particular a polyethylene glycol (PEG)
moiety. Antibody-like proteins may comprise a cysteine containing
linker, such as the linker set forth in SEQ ID NO: 10, 81, 97 or
218. PEG may be conjugated to the cysteine moiety in the linker
sequence and may operably link the two domains.
Pharmacokinetic Moieties
In one aspect, the disclosure provides E binders and E/I binders
further comprising a pharmacokinetic (PK) moiety. In some
embodiments, the E/I binder is a multimer of antibody-like
proteins, in particular, a dimer of an EGFR binding .sup.10Fn3 and
an IGFIR binding .sup.10Fn3. Improved pharmacokinetics may be
assessed according to the perceived therapeutic need. Often it is
desirable to increase bioavailability and/or increase the time
between doses, possibly by increasing the time that a protein
remains available in the serum after dosing. In some instances, it
is desirable to improve the continuity of the serum concentration
of the protein over time (e.g., decrease the difference in serum
concentration of the protein shortly after administration and
shortly before the next administration). E binders and E/I binders
may be attached to a moiety that reduces the clearance rate of the
polypeptide in a mammal (e.g., mouse, rat, or human) by greater
than three-fold relative to the unmodified polypeptide. Other
measures of improved pharmacokinetics may include serum half-life,
which is often divided into an alpha phase and a beta phase. Either
or both phases may be improved significantly by addition of an
appropriate moiety.
Moieties that tend to slow clearance of a protein from the blood
include polyoxyalkylene moieties (e.g., polyethylene glycol);
sugars (e.g., sialic acid); and well-tolerated protein moieties
(e.g., Fc, Fc fragments, transferrin, or serum albumin).
In some embodiments, the PK moiety is a serum albumin binding
protein such as those described in U.S. Publication Nos.
2007/0178082 and 2007/0269422.
In some embodiments, the PK moiety is a serum immunoglobulin
binding protein such as those described in U.S. Publication No.
2007/0178082.
In some embodiments, the PK moiety is polyethylene glycol
(PEG).
The serum clearance rate of a PK-modified antibody-like protein
multimer may be decreased by about 10%, 20%, 30%, 40%, 50%, 60%,
70%, 80%, or even 90%, relative to the clearance rate of the
unmodified E/I binders. The PK-modified multimer may have a
half-life (t.sub.1/2) which is enhanced relative to the half-life
of the unmodified multimer. The half-life of PK-binding polypeptide
may be enhanced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,
90%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 400% or 500%, or
even by 1000% relative to the half-life of the unmodified multimer.
In some embodiments, the multimer half-life is determined in vitro,
such as in a buffered saline solution or in serum. In other
embodiments, the multimer half-life is an in vivo half life, such
as the half-life of the multimer in the serum or other bodily fluid
of an animal.
In some embodiments, a PK moiety is linked to an antibody-like
protein multimer via at least one disulfide bond, a peptide bond, a
polypeptide, a polymeric sugar, or a polyethylene glycol moiety.
Exemplary polypeptide linkers include PSTSTST (SEQ ID NO: 12),
EIDKPSQ (SEQ ID NO: 9), and GS linkers, such as GSGSGSGSGS (SEQ ID
NO: 13) and multimers thereof.
Binding/Screening
The disclosure provides E binders and E/I binders, in particular,
antibody-like protein multimers such as a dimer of an EGFR binding
.sup.10Fn3 and an IGFIR binding .sup.10Fn3. Binding to EGFR or
IGFIR may be assessed in terms of equilibrium constants (e.g.,
dissociation, K.sub.D) and in terms of kinetic constants (e.g., on
rate constant, k.sub.on and off rate constant, k.sub.off). In some
embodiments, an antibody-like protein monomer or multimer will bind
to EGFR with a K.sub.D of less than 500 nM, 100 nM, 50 nM, 5 nM or
less. In some embodiments, an antibody-like protein multimer will
bind to IGFIR with a K.sub.D of less than 500 nM, 100 nM, 50 nM, 5
nM or less. Higher K.sub.D values may be tolerated where the
k.sub.off is sufficiently low or the k.sub.on is sufficiently
high.
E binders and E/I binders may bind to any part of EGFR, including
the extracellular domain of a EGFR, in particular the ligand
binding domain of EGFR. Binding of E binders and E/I binders to
EGFR may disrupt the interaction of EGFR with one or more ligands,
including TGF-alpha and EGF, and/or disrupt receptor dimerization.
In some embodiments, E binders and E/I binders compete with an
anti-EGFR antibody for binding to EGFR. The anti-EGFR antibody may
be selected from any known anti-EGFR antibody including panitumumab
(Amgen), nimotuzumab (YM Biosciences), zalutumumab (Genmab),
EMD72000 (Merck KGaA), and cetuximab (ImClone Systems).
In some embodiments, E binders and E/I binders inhibit downstream
signaling of EGFR. EGFR ligand binding leads to homo- or
heterodimeric receptor dimerization with EGFR or another HER family
member. Dimerization promotes receptor autophosphorylation, which
in turn leads to the activation of several signaling pathways.
E/I binders may bind to any part of IGFIR, including the
extracellular domain of a IGFIR, in particular the ligand binding
domain of IGFIR. Binding of E/I binders to IGFIR may disrupt the
interaction of IGFIR with one or more ligands, e.g., IGF-I and
IGF-II; and/or disrupt assembly of receptor heterotetramers. In
some embodiments, E/I binders compete with an anti-IGFIR antibody
for binding to IGFIR. The anti-IGFIR antibody may be selected from
any known anti-IGFIR antibody.
In some embodiments, E/I binders inhibit downstream signaling of
IGFIR. The IGF-I receptor is composed of two types of subunits: an
alpha subunit (a 130-135 kDa protein that is entirely extracellular
and functions in ligand binding) and a beta subunit (a 95-kDa
transmembrane protein, with transmembrane and cytoplasmic domains).
IGFIR is initially synthesized as a single chain proreceptor
polypeptide that is processed by glycosylation, proteolytic
cleavage, and covalent bonding to assemble into a mature 460-kDa
heterotetramer comprising two alpha-subunits and two beta-subunits.
The beta subunit(s) possesses ligand-activated tyrosine kinase
activity.
EGFR and IGFIR receptor signaling independently activates the MAPK
pathway, including the phosphorylation of MEK. Another activated
pathway is the phosphatidylinositol 3-kinase (PI3K) pathway,
including phosphorylation of AKT. Receptor signaling is transduced
to the nucleus, resulting in the activation of various
transcription factors.
Screening assays may be designed to identify and characterize E
binders and E/I binders. Binding assays, such as surface plasmon
resonance and ELISA, and assays that detect activated signaling
pathways are well-known in the art, see e.g., Example 5. Various
antibodies, including many that are commercially available, have
been produced which specifically bind to phosphorylated, activated
isoforms of EGFR and IGFIR, see e.g., Examples 6 and 7. Downstream
signaling events may also be used as an indicator of receptor
inhibition, such as by measuring levels of AKT phosphorylation, see
e.g., Example 8. Cell proliferation assays are also a useful method
for characterizing the ability of candidate E/I binders to bind and
inhibit EGFR and IGFIR signaling, see e.g., Example 9.
Polymer Conjugation
Conjugation to a biocompatible polymer may be used to link
antibody-like protein multimers and/or to improve the
pharmacokinetics of the proteins. The identity, size and structure
of the polymer is selected so as to improve the circulation
half-life of the multimer or decrease the antigenicity of the
multimer without an unacceptable decrease in activity.
Examples of polymers useful in the invention include, but are not
limited to, poly(alkylene glycols) such as polyethylene glycol
(PEG). The polymer is not limited to a particular structure and can
be linear (e.g., alkoxy PEG or bifunctional PEG), or non-linear
such as branched, forked, multi-armed (e.g., PEGs attached to a
polyol core), and dendritic.
Typically, PEG and other water-soluble polymers (i.e., polymeric
reagents) are activated with a suitable activating group
appropriate for coupling to a desired site on the polypeptide.
Thus, a polymeric reagent will possess a reactive group for
reaction with the polypeptide. Representative polymeric reagents
and methods for conjugating these polymers to an active moiety are
well-known in the art and further described in Zalipsky, S., et
al., "Use of Functionalized Poly(Ethylene Glycols) for Modification
of Polypeptides" in Polyethylene Glycol Chemistry: Biotechnical and
Biomedical Applications, J. M. Harris, Plenus Press, New York
(1992), and in Zalipsky (1995) Advanced Drug Reviews 16:
157-182.
Typically, the weight-average molecular weight of the polymer is
from about 100 Daltons to about 150,000 Daltons. Exemplary
weight-average molecular weights for the biocompatible polymer
include about 20,000 Daltons, about 40,000 Daltons, about 60,000
Daltons and about 80,000 Daltons. Branched versions of the
biocompatible polymer having a total molecular weight of any of the
foregoing can also be used.
In some embodiments, the polymer is PEG. PEG is a well-known, water
soluble polymer that is commercially available or can be prepared
by ring-opening polymerization of ethylene glycol according to
methods well known in the art (Sandler and Karo, Polymer Synthesis,
Academic Press, New York, Vol. 3, pages 138-161). The term "PEG" is
used broadly to encompass any polyethylene glycol molecule, without
regard to size or to modification at an end of the PEG, and can be
represented by the formula:
X--O(CH.sub.2CH.sub.2O).sub.n-1CH.sub.2CH.sub.2OH, where n is 20 to
2300 and X is H or a terminal modification, e.g., a C.sub.1-4
alkyl. PEG can contain further chemical groups which are necessary
for binding reactions, which result from the chemical synthesis of
the molecule; or which act as a spacer for optimal distance of
parts of the molecule. In addition, such a PEG can consist of one
or more PEG side-chains which are linked together. PEGs with more
than one PEG chain are called multiarmed or branched PEGs. Branched
PEG are described in, for example, European Published Application
No. 473084A and U.S. Pat. No. 5,932,462.
To effect covalent attachment of the polymer molecule(s) to a
polypeptide, the hydroxyl end groups of the polymer molecule must
be provided in activated form, i.e. with reactive functional
groups. Suitably activated polymer molecules are commercially
available, e.g. from Nektar Therapeutics, Inc., Huntsville, Ala.,
USA; PolyMASC Pharmaceuticals plc, UK; or SunBio Corporation,
Anyang City, South Korea. Alternatively, the polymer molecules can
be activated by conventional methods known in the art, e.g. as
disclosed in WO 90/13540. Specific examples of activated PEG
polymers include the following linear PEGs: NHS-PEG, SPA-PEG,
SSPA-PEG, SBA-PEG, SS-PEG, SSA-PEG, SC-PEG, SG-PEG, SCM-PEG,
NOR-PEG, BTC-PEG, EPDX-PEG, NCO-PEG, NPC-PEG, CDI-PEG, ALD-PEG,
TRES-PEG, VS-PEG, OPSSu-PEG, IODO-PEG, and MAL-PEG, and branched
PEGs, such as PEG2-NHS, PEG2-MAL, and those disclosed in U.S. Pat.
Nos. 5,932,462 and 5,643,575, both of which are incorporated herein
by reference.
In some embodiments where PEG molecules are conjugated to cysteine
residues on an antibody-like protein multimer, the cysteine
residues are native to the protein, whereas in other embodiments,
one or more cysteine residues are engineered into the protein.
Mutations may be introduced into a protein coding sequence to
generate cysteine residues. This might be achieved, for example, by
mutating one or more amino acid residues to cysteine. Preferred
amino acids for mutating to a cysteine residue include serine,
threonine, alanine and other hydrophilic residues. Preferably, the
residue to be mutated to cysteine is a surface-exposed residue.
Algorithms are well-known in the art for predicting surface
accessibility of residues based on primary sequence or a protein.
Alternatively, surface residues may be predicted by comparing the
amino acid sequences of binding polypeptides, given that the
crystal structure of the framework based on which binding
polypeptides are designed and evolved has been solved (see Himanen
et al., Nature. (2001) 20-27; 414(6866):933-8) and thus the
surface-exposed residues identified. In some embodiments, cysteine
residues are introduced into antibody-like protein multimers at or
near the N- and/or C-terminus, or within loop regions. Pegylation
of cysteine residues may be carried out using, for example,
PEG-maleimide, PEG-vinylsulfone, PEG-iodoacetamide, or
PEG-orthopyridyl disulfide.
In some embodiments, the pegylated antibody-like protein multimer
comprises a PEG molecule covalently attached to the alpha amino
group of the N-terminal amino acid. Site specific N-terminal
reductive amination is described in Pepinsky et al., (2001) JPET,
297,1059, and U.S. Pat. No. 5,824,784. The use of a PEG-aldehyde
for the reductive amination of a protein utilizing other available
nucleophilic amino groups is described in U.S. Pat. No. 4,002,531,
in Wieder et al., (1979) J. Biol. Chem. 254, 12579, and in Chamow
et al., (1994) Bioconjugate Chem. 5, 133.
In another embodiment, pegylated antibody-like protein multimer
comprises one or more PEG molecules covalently attached to a
linker, which in turn is attached to the alpha amino group of the
amino acid residue at the N-terminus of the binding polypeptide.
Such an approach is disclosed in U.S. Publication No. 2002/0044921
and PCT Publication No. WO 94/01451.
In some embodiments, an antibody-like protein multimer is pegylated
at the C-terminus A protein may be pegylated at the C-terminus by
the introduction of C-terminal azido-methionine and the subsequent
conjugation of a methyl-PEG-triarylphosphine compound via the
Staudinger reaction. This C-terminal conjugation method is
described in Cazalis et al., C-Terminal Site-Specific PEGylation of
a Truncated Thrombomodulin Mutant with Retention of Full
Bioactivity, Bioconjug Chem. 2004; 15(5):1005-1009.
Conventional separation and purification techniques known in the
art can be used to purify PEGylated antibody-like protein
multimers, such as size exclusion (e.g., gel filtration) and ion
exchange chromatography. Products may also be separated using
SDS-PAGE. Products that may be separated include mono-, di-, tri-,
poly- and un-pegylated binding polypeptide, as well as free PEG.
The percentage of mono-PEG conjugates can be controlled by pooling
broader fractions around the elution peak to increase the
percentage of mono-PEG in the composition. About ninety percent
mono-PEG conjugates represents a good balance of yield and
activity.
In some embodiments, the pegylated antibody-like protein multimers
will preferably retain at least about 25%, 50%, 60%, 70%, 80%, 85%,
90%, 95% or 100% of the biological activity associated with the
unmodified protein. In some embodiments, biological activity refers
to its ability to bind to EGFR and IGFIR, as assessed by K.sub.D,
k.sub.on or k.sub.off. In some embodiments, the pegylated
antibody-like protein multimer shows an increase in binding to EGFR
and/or IGFIR relative to unpegylated protein.
Deimmunization of Binding Polypeptides
The amino acid sequences of E binders and E/I binders, in
particular, antibody-like protein multimers, such as a dimer of an
EGFR binding .sup.10Fn3 and an IGFIR binding .sup.10Fn3, may be
altered to eliminate one or more B- or T-cell epitopes. A protein,
or a multimer of proteins, may be deimmunized to render it
non-immunogenic, or less immunogenic, to a given species.
Deimmunization can be achieved through structural alterations to
the protein. Any deimmunization technique known to those skilled in
the art can be employed, see e.g., WO 00/34317, the disclosure of
which is incorporated herein in its entirety.
In one embodiment, the sequences of the E binders and E/I binders
can be analyzed for the presence of MHC class II binding motifs.
For example, a comparison may be made with databases of MHC-binding
motifs such as, for example by searching the "motifs" database on
the worldwide web at sitewehil.wehi.edu.au. Alternatively, MHC
class II binding peptides may be identified using computational
threading methods such as those devised by Altuvia et al. (J. Mol.
Biol. 249 244-250 (1995)) whereby consecutive overlapping peptides
from the polypeptide are testing for their binding energies to MHC
class II proteins. Computational binding prediction algorithms
include iTope.TM., Tepitope, SYFPEITHI, EpiMatrix (EpiVax), and
MHCpred. In order to assist the identification of MHC class
II-binding peptides, associated sequence features which relate to
successfully presented peptides such as amphipathicity and Rothbard
motifs, and cleavage sites for cathepsin B and other processing
enzymes can be searched for.
Having identified potential (e.g. human) T-cell epitopes, these
epitopes are then eliminated by alteration of one or more amino
acids, as required to eliminate the T-cell epitope. Usually, this
will involve alteration of one or more amino acids within the
T-cell epitope itself. This could involve altering an amino acid
adjacent the epitope in terms of the primary structure of the
protein or one which is not adjacent in the primary structure but
is adjacent in the secondary structure of the molecule. The usual
alteration contemplated will be amino acid substitution, but it is
possible that in certain circumstances amino acid addition or
deletion will be appropriate. All alterations can be accomplished
by recombinant DNA technology, so that the final molecule may be
prepared by expression from a recombinant host, for example by well
established methods, but the use of protein chemistry or any other
means of molecular alteration may also be used.
Once identified T-cell epitopes are removed, the deimmunized
sequence may be analyzed again to ensure that new T-cell epitopes
have not been created and, if they have, the epitope(s) can be
deleted.
Not all T-cell epitopes identified computationally need to be
removed. A person skilled in the art will appreciate the
significance of the "strength" or rather potential immunogenicity
of particular epitopes. The various computational methods generate
scores for potential epitopes. A person skilled in the art will
recognize that only the high scoring epitopes may need to be
removed. A skilled person will also recognize that there is a
balance between removing potential epitopes and maintaining binding
affinity of the protein. Therefore, one strategy is to sequentially
introduce substitutions into the protein and then test for antigen
binding and immunogenicity.
In one aspect, the deimmunized protein is less immunogenic (or
rather, elicits a reduced HAMA response) than the original protein
in a human subject. Assays to determine immunogenicity are well
within the knowledge of the skilled person. Art-recognized methods
of determining immune response can be performed to monitor a HAMA
response in a particular subject or during clinical trials.
Subjects administered deimmunized protein can be given an
immunogenicity assessment at the beginning and throughout the
administration of said therapy. The HAMA response is measured, for
example, by detecting antibodies to the deimmunized protein in
serum samples from the subject using a method known to one in the
art, including surface plasmon resonance technology (BIAcore)
and/or solid-phase ELISA analysis. Alternatively, in vitro assays
designed to measure a T-cell activation event are also indicative
of immunogenicity.
Additional Modifications
In certain embodiments, E binders and E/I binders, in particular,
antibody-like protein multimers such as a dimer of an EGFR binding
.sup.10Fn3 and an IGFIR binding .sup.10Fn3, may further comprise
post-translational modifications. Exemplary post-translational
protein modification include phosphorylation, acetylation,
methylation, ADP-ribosylation, ubiquitination, glycosylation,
carbonylation, sumoylation, biotinylation or addition of a
polypeptide side chain or of a hydrophobic group. As a result, the
modified E binders and E/I binders may contain non-amino acid
elements, such as lipids, poly- or mono-saccharide, and phosphates.
A preferred form of glycosylation is sialylation, which conjugates
one or more sialic acid moieties to the polypeptide. Sialic acid
moieties improve solubility and serum half-life while also reducing
the possible immunogenicity of the protein. See, e.g., Raju et al.
Biochemistry. 2001 Jul. 31; 40(30):8868-76. Effects of such
non-amino acid elements on the functionality of an E binder or E/I
binder may be tested for its antagonizing role in EGFR and IGFIR
signaling function.
In some embodiments, E binders and E/I binders are modified to
enhance antigen-dependent cell-mediated cytotoxicity (ADCC) and/or
complement dependent cytotoxicity (CDC). In some embodiments, the
E/I binder is a dimer of an EGFR binding .sup.10Fn3 and an IGFIR
binding .sup.10Fn3, further comprising an Fc region. In some
embodiments, the Fc region is a variant that enhances ADCC or CDC.
The Fc region variant may comprise a human Fc region sequence
(e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an
amino acid modification (e.g., a substitution) at one or more amino
acid positions, including positions 256, 290, 298, 312, 326, 330,
333, 334, 360, 378 or 430, wherein the numbering of the residues in
the Fc region is that of the EU index as in Kabat.
Vectors & Polynucleotides Embodiments
Also included in the present disclosure are nucleic acid sequences
encoding any of the proteins described herein. As appreciated by
those skilled in the art, because of third base degeneracy, almost
every amino acid can be represented by more than one triplet codon
in a coding nucleotide sequence. In addition, minor base pair
changes may result in a conservative substitution in the amino acid
sequence encoded but are not expected to substantially alter the
biological activity of the gene product. Therefore, a nucleic acid
sequence encoding a protein described herein may be modified
slightly in sequence and yet still encode its respective gene
product.
Exemplary nucleic acids encoding the E/I binders described herein
include nucleic acids having SEQ ID NOs: 442-465 or nucleic acids
having a sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or
100% identical to any one of SEQ ID NOs: 442-465. Isolated nucleic
acids which differ from the nucleic acids as set forth in SEQ ID
NOs: 442-465 due to degeneracy in the genetic code are also within
the scope of the invention. Also provided are E/I binders
comprising an I monomer encoded by a nucleotide sequence at least
80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:
328 and/or E/I binders comprising an E monomer encoded by a
nucleotide sequence at 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%
identical to any one of SEQ ID NOs: 329-441 or 495. Also provided
are E binders encoded by a nucleotide sequence at 80%, 85%, 90%,
95%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs:
329-441 or 495. In certain embodiments, the nucleotide sequences
encoding the E/I binders, an E monomer, or an I monomer do not
contain a sequence encoding a 6.times.His tag (SEQ ID NO: 487).
Nucleic acids encoding any of the various proteins or polypeptides
disclosed herein may be synthesized chemically. Codon usage may be
selected so as to improve expression in a cell. Such codon usage
will depend on the cell type selected. Specialized codon usage
patterns have been developed for E. coli and other bacteria, as
well as mammalian cells, plant cells, yeast cells and insect cells.
See for example: Mayfield et al., Proc Natl Acad Sci USA. 2003
100(2):438-42; Sinclair et al. Protein Expr Purif. 2002 (1):96-105;
Connell N D. Curr Opin Biotechnol. 2001 (5):446-9; Makrides et al.
Microbiol Rev. 1996 60(3):512-38; and Sharp et al. Yeast. 1991
7(7):657-78.
General techniques for nucleic acid manipulation are within the
purview of one skilled in the art and are also described for
example in Sambrook et al., Molecular Cloning: A Laboratory Manual,
Vols. 1-3, Cold Spring Harbor Laboratory Press, 2 ed., 1989, or F.
Ausubel et al., Current Protocols in Molecular Biology (Green
Publishing and Wiley-Interscience: New York, 1987) and periodic
updates, herein incorporated by reference. The DNA encoding a
protein is operably linked to suitable transcriptional or
translational regulatory elements derived from mammalian, viral, or
insect genes. Such regulatory elements include a transcriptional
promoter, an optional operator sequence to control transcription, a
sequence encoding suitable mRNA ribosomal binding sites, and
sequences that control the termination of transcription and
translation. The ability to replicate in a host, usually conferred
by an origin of replication, and a selection gene to facilitate
recognition of transformants are additionally incorporated.
Suitable regulatory elements are well-known in the art.
The proteins described herein may be produced as a fusion protein
with a heterologous polypeptide, which is preferably a signal
sequence or other polypeptide having a specific cleavage site at
the N-terminus of the mature protein or polypeptide. The
heterologous signal sequence selected preferably is one that is
recognized and processed (i.e., cleaved by a signal peptidase) by
the host cell. For prokaryotic host cells that do not recognize and
process a native signal sequence, the signal sequence is
substituted by a prokaryotic signal sequence selected, for example,
from the group of the alkaline phosphatase, penicillinase, lpp, or
heat-stable enterotoxin II leaders. For yeast secretion, the native
signal sequence may be substituted by, e.g., the yeast invertase
leader, a factor leader (including Saccharomyces and Kluyveromyces
alpha-factor leaders), or acid phosphatase leader, the C. albicans
glucoamylase leader, or the signal described in PCT Publication No.
WO 90/13646. In mammalian cell expression, mammalian signal
sequences as well as viral secretory leaders, for example, the
herpes simplex gD signal, are available. The DNA for such precursor
regions may be ligated in reading frame to DNA encoding the
protein.
Expression vectors used in eukaryotic host cells (e.g., yeast,
fungi, insect, plant, animal, human, or nucleated cells from other
multicellular organisms) will also contain sequences necessary for
the termination of transcription and for stabilizing the mRNA. Such
sequences are commonly available from the 5' and, occasionally 3',
untranslated regions of eukaryotic or viral DNAs or cDNAs. These
regions contain nucleotide segments transcribed as polyadenylated
fragments in the untranslated portion of the mRNA encoding the
multivalent antibody. One useful transcription termination
component is the bovine growth hormone polyadenylation region. See
PCT Publication No. WO 94/11026 and the expression vector disclosed
therein.
The recombinant DNA can also include any type of protein tag
sequence that may be useful for purifying the protein. Examples of
protein tags include but are not limited to a histidine tag, a FLAG
tag, a myc tag, an HA tag, or a GST tag. Appropriate cloning and
expression vectors for use with bacterial, fungal, yeast, and
mammalian cellular hosts can be found in Cloning Vectors: A
Laboratory Manual, (Elsevier, New York, 1985), the relevant
disclosure of which is hereby incorporated by reference.
The expression construct is introduced into the host cell using a
method appropriate to the host cell, as will be apparent to one of
skill in the art. A variety of methods for introducing nucleic
acids into host cells are known in the art, including, but not
limited to, electroporation; transfection employing calcium
chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or
other substances; microprojectile bombardment; lipofection; and
infection (where the vector is an infectious agent).
Suitable host cells include prokaryotes, yeast, mammalian cells, or
bacterial cells. Suitable bacteria include gram negative or gram
positive organisms, for example, E. coli or Bacillus spp. Yeast,
preferably from the Saccharomyces species, such as S. cerevisiae,
may also be used for production of polypeptides. Various mammalian
or insect cell culture systems can also be employed to express
recombinant proteins. Baculovirus systems for production of
heterologous proteins in insect cells are reviewed by Luckow and
Summers, (Bio/Technology, 6:47, 1988). In some instance it will be
desired to produce proteins in vertebrate cells, such as for
glycosylation, and the propagation of vertebrate cells in culture
(tissue culture) has become a routine procedure. Examples of
suitable mammalian host cell lines include endothelial cells, COS-7
monkey kidney cells, CV-1, L cells, C127, 3T3, Chinese hamster
ovary (CHO), human embryonic kidney cells, HeLa, 293, 293T, and BHK
cell lines. For many applications, the small size of the protein
multimers described herein would make E. coli the preferred method
for expression.
Protein Production
Host cells are transformed with the herein-described expression or
cloning vectors for protein production and cultured in conventional
nutrient media modified as appropriate for inducing promoters,
selecting transformants, or amplifying the genes encoding the
desired sequences.
The host cells used to produce the proteins of this invention may
be cultured in a variety of media. Commercially available media
such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), Sigma),
RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM),
Sigma) are suitable for culturing the host cells. In addition, any
of the media described in Ham et al., Meth. Enz. 58:44 (1979),
Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Pat. Nos.
4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO
90/03430; WO 87/00195; or U.S. Pat. No. Re. 30,985 may be used as
culture media for the host cells. Any of these media may be
supplemented as necessary with hormones and/or other growth factors
(such as insulin, transferrin, or epidermal growth factor), salts
(such as sodium chloride, calcium, magnesium, and phosphate),
buffers (such as HEPES), nucleotides (such as adenosine and
thymidine), antibiotics (such as GENTAMYCIN.TM. drug), trace
elements (defined as inorganic compounds usually present at final
concentrations in the micromolar range), and glucose or an
equivalent energy source. Any other necessary supplements may also
be included at appropriate concentrations that would be known to
those skilled in the art. The culture conditions, such as
temperature, pH, and the like, are those previously used with the
host cell selected for expression, and will be apparent to the
ordinarily skilled artisan.
Proteins disclosed herein can also be produced using
cell-translation systems. For such purposes, the nucleic acids
encoding the proteins must be modified to allow in vitro
transcription to produce mRNA and to allow cell-free translation of
the mRNA in the particular cell-free system being utilized.
Exemplary eukaryotic cell-free translation systems include, for
example, mammalian or yeast cell-free translation systems, and
exemplary prokaryotic cell-free translation systems include, for
example, bacterial cell-free translation systems.
Proteins disclosed herein can also be produced by chemical
synthesis (e.g., by the methods described in Solid Phase Peptide
Synthesis, 2nd ed., 1984, The Pierce Chemical Co., Rockford, Ill.).
Modifications to the protein can also be produced by chemical
synthesis.
The proteins disclosed herein can be purified by
isolation/purification methods for proteins generally known in the
field of protein chemistry. Non-limiting examples include
extraction, recrystallization, salting out (e.g., with ammonium
sulfate or sodium sulfate), centrifugation, dialysis,
ultrafiltration, adsorption chromatography, ion exchange
chromatography, hydrophobic chromatography, normal phase
chromatography, reversed-phase chromatography, gel filtration, gel
permeation chromatography, affinity chromatography,
electrophoresis, countercurrent distribution or any combinations of
these. After purification, proteins may be exchanged into different
buffers and/or concentrated by any of a variety of methods known to
the art, including, but not limited to, filtration and
dialysis.
The purified proteins are preferably at least 85% pure, more
preferably at least 95% pure, and most preferably at least 98%
pure. Regardless of the exact numerical value of the purity, the
proteins are sufficiently pure for use as a pharmaceutical
product.
Imaging, Diagnostic and Other Applications
The E binders described herein can be detectably labeled and used
to contact cells expressing EGFR for imaging or diagnostic
applications. The E/I binders described herein can be detectably
labeled and used to contact cells expressing EGFR and/or IGFIR for
imaging or diagnostic applications. Any method known in the art for
conjugating a protein to the detectable moiety may be employed,
including those methods described by Hunter, et al., Nature 144:945
(1962); David, et al., Biochemistry 13:1014 (1974); Pain, et al.,
J. Immunol. Meth. 40:219 (1981); and Nygren, J. Histochem. and
Cytochem. 30:407 (1982).
In certain embodiments, the E binders and E/I binders described
herein are further attached to a label that is able to be detected
(e.g., the label can be a radioisotope, fluorescent compound,
enzyme or enzyme co-factor). The label may be a radioactive agent,
such as: radioactive heavy metals such as iron chelates,
radioactive chelates of gadolinium or manganese, positron emitters
of oxygen, nitrogen, iron, carbon, or gallium, .sup.43K, .sup.52Fe,
.sup.57Co, .sup.67Cu, .sup.67Ga, .sup.68Ga, .sup.123I, .sup.125I,
.sup.131I, .sup.132I, or .sup.99Tc. An E binder or E/I binder
affixed to such a moiety may be used as an imaging agent and is
administered in an amount effective for diagnostic use in a mammal
such as a human and the localization and accumulation of the
imaging agent is then detected. The localization and accumulation
of the imaging agent may be detected by radioscintigraphy, nuclear
magnetic resonance imaging, computed tomography or positron
emission tomography. As will be evident to the skilled artisan, the
amount of radioisotope to be administered is dependent upon the
radioisotope. Those having ordinary skill in the art can readily
formulate the amount of the imaging agent to be administered based
upon the specific activity and energy of a given radionuclide used
as the active moiety.
E binders and E/I binders also are useful as affinity purification
agents. In this process, the proteins are immobilized on a suitable
support, such a Sephadex resin or filter paper, using methods well
known in the art. The proteins can be employed in any known assay
method, such as competitive binding assays, direct and indirect
sandwich assays, and immunoprecipitation assays (Zola, Monoclonal
Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc.,
1987)).
E binders are useful in methods for detecting EGFR in a sample. E/I
binders also are useful in methods for detecting EGFR and/or IGFIR
in a sample. The sample will often by a biological sample, such as
a biopsy, and particularly a biopsy of a tumor, a suspected tumor.
The sample may be from a human or other mammal. The E binder or E/I
binder may be labeled with a labeling moiety, such as a radioactive
moiety, a fluorescent moiety, a chromogenic moiety, a
chemiluminescent moiety, or a hapten moiety; and may be immobilized
on a solid support. Detection may be carried out using any
technique known in the art, such as, for example, radiography,
immunological assay, fluorescence detection, mass spectroscopy, or
surface plasmon resonance.
Therapeutic/In Vivo Uses
The E binders described herein are also useful in methods for
treating conditions which respond to an inhibition of EGFR
biological activity. The E/I binders described herein are also
useful in methods for treating conditions which respond to an
inhibition of EGFR and/or IGFIR biological activity. EGFR and IGFIR
are involved either directly or indirectly in the signal
transduction pathways of various cell activities, including
proliferation, adhesion and migration, as well as
differentiation.
In one aspect, the application provides methods for treating a
subject afflicted with a hyperproliferative disorder with a
therapeutically effective amount of an E binder or an E/I binder.
In particular, E binders and E/I binders are useful for the
treatment and/or prophylaxis of tumors and/or tumor metastases. In
exemplary embodiments, the E/I binder is an antibody-like protein
multimer such as a dimer of an EGFR binding .sup.10Fn3 and an IGFIR
binding .sup.10Fn3.
In some embodiments, pharmaceutical compositions comprising E
binders or E/I binders are administered to a subject afflicted with
a tumor, including but not limited to, a brain tumor, tumor of the
urogenital tract, tumor of the lymphatic system, stomach tumor,
laryngeal tumor, monocytic leukemia, lung adenocarcinoma,
small-cell lung carcinoma, pancreatic cancer, glioblastoma and
breast carcinoma; or a cancerous disease, including but not limited
to, squamous cell carcinoma, bladder cancer, stomach cancer, liver
cancer, kidney cancer, colorectal cancer, breast cancer, head
cancer, neck cancer, oesophageal cancer, gynecological cancer,
thyroid cancer, lymphoma, chronic leukemia and acute leukemia.
An E binder or an E/I binder can be administered alone or in
combination with one or more additional therapies such as
chemotherapy radiotherapy, immunotherapy, surgical intervention, or
any combination of these. Long-term therapy is equally possible as
is adjuvant therapy in the context of other treatment strategies,
as described herein. Techniques and dosages for administration vary
depending on the type of specific polypeptide and the specific
condition being treated but can be readily determined by the
skilled artisan.
Additional Agents that May be Used with E/I Binders
One aspect of the application provides combinations of E binder or
E/I binders and an additional therapeutic agent, such as a
cytotoxic agent. In some embodiments, an E binder or E/I binder is
linked to a cytotoxic agent. Such embodiments can be prepared by in
vitro or in vivo methods as appropriate. In vitro methods include
conjugation chemistry well know in the art, such as conjugation to
cysteine and lysine residues. In order to link a cytotoxic agent to
a polypeptide, a linking group or reactive group is used. Suitable
linking groups are well known in the art and include disulfide
groups, thioether groups, acid labile groups, photolabile groups,
peptidase labile groups and esterase labile groups. Cytotoxic
agents can also be linked to E binders or E/I binders through an
intermediary carrier molecule such as serum albumin
Exemplary cytotoxic agents that may be linked to E binders or E/I
binders, include maytansinoids, taxanes, analogs of CC-1065,
bacterial toxin, plant toxin, ricin, abrin, a ribonuclease (RNase),
DNase I, a protease, Staphylococcal enterotoxin-A, pokeweed
antiviral protein, gelonin, diphtherin toxin, Pseudomonas exotoxin,
Pseudomonas endotoxin, Ranpimase (Rap), Rap (N69Q), methotrexate,
daunorubicin, doxorubicin, vincristine, vinblastine, melphalan,
mitomycin C, chlorambucil, and calicheamicin.
In other therapeutic treatments or compositions, E binders or E/I
binders are co-administered, or administered sequentially, with one
or more additional therapeutic agents. Suitable therapeutic agents
include, but are not limited to, cytotoxic or cytostatic agents,
such as cancer therapeutic agents.
Cancer therapeutic agents are those agents that seek to kill or
limit the growth of cancer cells while having minimal effects on
the patient. Thus, such agents may exploit any difference in cancer
cell properties (e.g., metabolism, vascularization or cell-surface
antigen presentation) from healthy host cells. Therapeutic agents
that can be combined with E/I binders for improved anti-cancer
efficacy include diverse agents used in oncology practice
(Reference: Cancer, Principles & Practice of Oncology, DeVita,
V. T., Hellman, S., Rosenberg, S. A., 6th edition,
Lippincott-Raven, Philadelphia, 2001), such as doxorubicin,
epirubicin, cyclophosphamide, trastuzumab, capecitabine, tamoxifen,
toremifene, letrozole, anastrozole, fulvestrant, exemestane,
goserelin, oxaliplatin, carboplatin, cisplatin, dexamethasone,
antide, bevacizumab, 5-fluorouracil, leucovorin, levamisole,
irinotecan, etoposide, topotecan, gemcitabine, vinorelbine,
estramustine, mitoxantrone, abarelix, zoledronate, streptozocin,
rituximab, idarubicin, busulfan, chlorambucil, fludarabine,
imatinib, cytarabine, ibritumomab, to situmomab, interferon
alpha-2b, melphalam, bortezomib, altretamine, asparaginase,
gefitinib, erlonitib, anti-EGF receptor antibody (e.g., cetuximab
or panitumab), ixabepilone, epothilones or derivatives thereof,
platinum agents (such as carboplatin, oxaliplatin, cisplatin),
taxanes (such as paclitaxel, docetaxel), and camptothecin.
Therapeutic Formulations and Modes of Administration
The present application provides methods for treating conditions
which respond to an inhibition of EGFR and/or IGFIR biological
activity. Techniques and dosages for administration vary depending
on the type of specific polypeptide and the specific condition
being treated but can be readily determined by the skilled artisan.
In general, regulatory agencies require that a protein reagent to
be used as a therapeutic is formulated so as to have acceptably low
levels of pyrogens. Accordingly, therapeutic formulations will
generally be distinguished from other formulations in that they are
substantially pyrogen free, or at least contain no more than
acceptable levels of pyrogen as determined by the appropriate
regulatory agency (e.g., FDA).
In some embodiments, the E binders and E/I binders are
pharmaceutically acceptable to a mammal, in particular a human. A
"pharmaceutically acceptable" polypeptide refers to a polypeptide
that is administered to an animal without significant adverse
medical consequences. Examples of pharmaceutically acceptable E
binders and E/I binders include .sup.10Fn3 domains that lack the
integrin-binding domain (RGD) and .sup.10Fn3 domains that are
essentially endotoxin free or have very low endotoxin levels.
Therapeutic compositions may be administered with a
pharmaceutically acceptable diluent, carrier, or excipient, in unit
dosage form. Administration may be parenteral (e.g., intravenous,
subcutaneous), oral, or topical, as non-limiting examples. In
addition, any gene therapy technique using nucleic acids encoding E
binders or E/I binders, may be employed, such as naked DNA
delivery, recombinant genes and vectors, cell-based delivery,
including ex vivo manipulation of patients' cells, and the
like.
The composition can be in the form of a pill, tablet, capsule,
liquid, or sustained release tablet for oral administration; a
liquid for intravenous, subcutaneous or parenteral administration;
or a gel, lotion, ointment, cream, or a polymer or other sustained
release vehicle for local administration.
Methods well known in the art for making formulations are found,
for example, in "Remington: The Science and Practice of Pharmacy"
(20th ed., ed. A. R. Gennaro AR., 2000, Lippincott Williams &
Wilkins, Philadelphia, Pa.). Formulations for parenteral
administration may, for example, contain excipients, sterile water,
saline, polyalkylene glycols such as polyethylene glycol, oils of
vegetable origin, or hydrogenated napthalenes. Biocompatible,
biodegradable lactide polymer, lactide/glycolide copolymer, or
polyoxyethylene-polyoxypropylene copolymers may be used to control
the release of the compounds. Nanoparticulate formulations (e.g.,
biodegradable nanoparticles, solid lipid nanoparticles, liposomes)
may be used to control the biodistribution of the compounds. Other
potentially useful parenteral delivery systems include
ethylene-vinyl acetate copolymer particles, osmotic pumps,
implantable infusion systems, and liposomes. The concentration of
the compound in the formulation varies depending upon a number of
factors, including the dosage of the drug to be administered, and
the route of administration.
The polypeptide may be optionally administered as a
pharmaceutically acceptable salt, such as non-toxic acid addition
salts or metal complexes that are commonly used in the
pharmaceutical industry. Examples of acid addition salts include
organic acids such as acetic, lactic, pamoic, maleic, citric,
malic, ascorbic, succinic, benzoic, palmitic, suberic, salicylic,
tartaric, methanesulfonic, toluenesulfonic, or trifluoroacetic
acids or the like; polymeric acids such as tannic acid,
carboxymethyl cellulose, or the like; and inorganic acid such as
hydrochloric acid, hydrobromic acid, sulfuric acid phosphoric acid,
or the like. Metal complexes include zinc, iron, and the like. In
one example, the polypeptide is formulated in the presence of
sodium acetate to increase thermal stability.
Formulations for oral use include tablets containing the active
ingredient(s) in a mixture with non-toxic pharmaceutically
acceptable excipients. These excipients may be, for example, inert
diluents or fillers (e.g., sucrose and sorbitol), lubricating
agents, glidants, and anti-adhesives (e.g., magnesium stearate,
zinc stearate, stearic acid, silicas, hydrogenated vegetable oils,
or talc).
Formulations for oral use may also be provided as chewable tablets,
or as hard gelatin capsules wherein the active ingredient is mixed
with an inert solid diluent, or as soft gelatin capsules wherein
the active ingredient is mixed with water or an oil medium.
A therapeutically effective dose refers to a dose that produces the
therapeutic effects for which it is administered. The exact dose
will depend on the disorder to be treated, and may be ascertained
by one skilled in the art using known techniques. In general, the E
binder or E/I binder is administered at about 0.01 mg/kg to about
50 mg/kg per day, preferably 0.01 mg/kg to about 30 mg/kg per day,
most preferably 0.1 mg/kg to about 20 mg/kg per day. The
polypeptide may be given daily (e.g., once, twice, three times, or
four times daily) or less frequently (e.g., once every other day,
once or twice weekly, or monthly). In addition, as is known in the
art, adjustments for age as well as the body weight, general
health, sex, diet, time of administration, drug interaction, and
the severity of the disease may be necessary, and will be
ascertainable with routine experimentation by those skilled in the
art.
EXEMPLIFICATION
The invention now being generally described will be more readily
understood by reference to the following examples which are
included merely for purposes of illustration of certain aspects and
embodiments of the present invention, and are not intended to limit
the invention in any way.
Summary of Sequences
Many of the sequences referenced in this application are summarized
in the table below. Unless otherwise specified, N-terminal
extensions are indicated with a single underline, C-terminal tails
are indicated with a double underline, and linker sequences are
indicated in bold.
TABLE-US-00011 SEQ ID NO: Description Sequence 1 WT human
.sup.10Fn3 domain VSDVPRDLEVVAATPTSLLISWDAPAVTVRYY
RITYGETGGNSPVQEFTVPGSKSTATISGLKPGV DYTITVYAVTGRGDSPASSKPISINYRT 2
Variant human .sup.10Fn3 with the VSDVPRDLEVVAATPTSLLISWDAPAVTVRYY
integrin binding motif removed RITYGETGGNSPVQEFTVPGSKSTATISGLKPGV
(RGD changed to SGE; DYTITVYAVTGSGESPASSKPISINYRT changes from SEQ
ID NO: 1 are underlined) 3 I1 IGF-IR monomer with N-
VSDVPRDLEVVAATPTSLLISWSARLKVARYY terminal extension (N + 8) and
RITYGETGGNSPVQEFTVPKNVYTATISGLKPG no tail VDYTITVYAVTRFRDYQPISINYRT
4 I1 IGF-IR monomer with MGVSDVPRDLEVVAATPTSLLISWSARLKVAR
N-terminal extension (N + 10) YYRITYGETGGNSPVQEFTVPKNVYTATISGLK and
Ser tail with His tag PGVDYTITVYAVTRFRDYQPISINYRTEIDKPSQ HHHHHH 5
E2 EGFR monomer with VSDVPRDLEVVAATPTSLLISWDSGRGSYQYY N-terminal
extension (N + 8) RITYGETGGNSPVQEFTVPGPVHTATISGLKPG and no tail
VDYTITVYAVTDHKPHADGPHTYHESPISINYR T 6 E2 EGFR monomer with
MGVSDVPRDLEVVAATPTSLLISWDSGRGSYQ N-terminal extension (N + 10)
YYRITYGETGGNSPVQEFTVPGPVHTATISGLK and Ser tail with his tag
PGVDYTITVYAVTDHKPHADGPHTYHESPISIN YRTEIDKPSQHHHHHH 7 E1 EGFR
monomer with VSDVPRDLEVVAATPTSLLISWVAGAEDYQYY N-terminal extension
(N + 8) RITYGETGGNSPVQEFTVPHDLVTATISGLKPG and no tail
VDYTITVYAVTDMMHVEYTEHPISINYRT 8 E1 EGFR monomer with
MGVSDVPRDLEVVAATPTSLLISWVAGAEDYQ N-terminal extension (N + 10)
YYRITYGETGGNSPVQEFTVPHDLVTATISGLK and Ser tail with his tag
PGVDYTITVYAVTDMMHVEYTEHPISINYRTEI DKPSQHHHHHH 9 Ser tail EIDKPSQ 10
Cys tail EIDKPCQ 11 (GS).sub.10 Linker GSGSGSGSGSGSGSGSGSGS 12 Fn
Based Linker PSTSTST 13 (GS).sub.5 Linker GSGSGSGSGS 14
(GGGGS).sub.3 Linker GGGGS GGGGS GGGGS 15 (GGGGS).sub.4 Linker
GGGGS GGGGS GGGGS GGGGS 16 (GGGGS).sub.5 Linker GGGGS GGGGS GGGGS
GGGGS GGGGS 17 G.sub.4SG.sub.4SG.sub.3SG Linker GGGGS GGGGS GGGSG
18 Linker GPGPGPG 19 Linker GPGPGPGPGPG 20 I1-GS10-E2: I/E tandem
VSDVPRDLEVVAATPTSLLISWSARLKVARYY having I1 (with N-terminal
RITYGETGGNSPVQEFTVPKNVYTATISGLKPG extension (N + 8) and short tail)
VDYTITVYAVTRFRDYQPISINYRTEIDKGSGS fused via GS.sub.10 linker (GS10
is GSGSGSGSGSGSGSGSVSDVPRDLEVVAATPT SEQ ID NO: 11) to E2 (with
SLLISWDSGRGSYQYYRITYGETGGNSPVQEFT N-terminal extension (N + 8)
VPGPVHTATISGLKPGVDYTITVYAVTDHKPHA and no tail) DGPHTYHESPISINYRT 21
I1-GS10-E2: I/E tandem MGVSDVPRDLEVVAATPTSLLISWSARLKVAR having I1
(with N-terminal YYRITYGETGGNSPVQEFTVPKNVYTATISGLK extension (N +
10) and short PGVDYTITVYAVTRFRDYQPISINYRTEIDKGSG tail) fused via
GS.sub.10 linker SGSGSGSGSGSGSGSGSVSDVPRDLEVVAATP (GS10 is SEQ ID
NO: 11) to TSLLISWDSGRGSYQYYRITYGETGGNSPVQEF E2 (with N-terminal
extension TVPGPVHTATISGLKPGVDYTITVYAVTDHKPH (N + 8) and Ser tail)
ADGPHTYHESPISINYRTEIDKPSQ 22 I1-GS10-E2: I/E tandem I1
MGVSDVPRDLEVVAATPTSLLISWSARLKVAR (with N-terminal extension
YYRITYGETGGNSPVQEFTVPKNVYTATISGLK (N + 10) and short tail) fused
PGVDYTITVYAVTRFRDYQPISINYRTEIDKGSG via GS.sub.10 linker (GS10 is
SEQ SGSGSGSGSGSGSGSGSVSDVPRDLEVVAATP ID NO: 11) to E2 (with N-
TSLLISWDSGRGSYQYYRITYGETGGNSPVQEF terminal extension (N + 8) and
TVPGPVHTATISGLKPGVDYTITVYAVTDHKPH Ser tail with his tag)
ADGPHTYHESPISINYRTEIDKPSQHHHHHH 23 E2-GS10-I1: E/I tandem
VSDVPRDLEVVAATPTSLLISWDSGRGSYQYY having E2 (with N-terminal
RITYGETGGNSPVQEFTVPGPVHTATISGLKPG extension (N + 8) and short tail)
VDYTITVYAVTDHKPHADGPHTYHESPISINYR fused via GS.sub.10 linker (GS10
is TEIDKGSGSGSGSGSGSGSGSGSGSVSDVPRD SEQ ID NO: 11) to I1 (with N-
LEVVAATPTSLLISWSARLKVARYYRITYGETG terminal extension (N + 8) and
GNSPVQEFTVPKNVYTATISGLKPGVDYTITVY Ser tail)
AVTRFRDYQPISINYRTEIDKPSQ 24 E2-GS10-I1: E/I tandem
MGVSDVPRDLEVVAATPTSLLISWDSGRGSYQ having E2 (with N-terminal
YYRITYGETGGNSPVQEFTVPGPVHTATISGLK extension (N + 10) and short
PGVDYTITVYAVTDHKPHADGPHTYHESPISIN tail) fused via GS.sub.10 linker
YRTEIDKGSGSGSGSGSGSGSGSGSGSVSDVP (GS10 is SEQ ID NO: 11) to
RDLEVVAATPTSLLISWSARLKVARYYRITYGE I1 (with N-terminal extension
TGGNSPVQEFTVPKNVYTATISGLKPGVDYTIT (N + 8) and Ser tail)
VYAVTRFRDYQPISINYRTEIDKPSQ 25 E2-GS10-I1: E/I tandem
MGVSDVPRDLEVVAATPTSLLISWDSGRGSYQ having E2 (with N-terminal
YYRITYGETGGNSPVQEFTVPGPVHTATISGLK extension (N + 10) and short
PGVDYTITVYAVTDHKPHADGPHTYHESPISIN tail) fused via GS.sub.10 linker
YRTEIDKGSGSGSGSGSGSGSGSGSGSVSDVP (GS10 is SEQ ID NO: 11) to
RDLEVVAATPTSLLISWSARLKVARYYRITYGE I1 (with N-terminal extension
TGGNSPVQEFTVPKNVYTATISGLKPGVDYTIT (N + 8) and Ser tail with his
tag) VYAVTRFRDYQPISINYRTEIDKPSQHHHHHH 26 I1-GS10-E1: I/E tandem
VSDVPRDLEVVAATPTSLLISWSARLKVARYY having I1 (with N-terminal
RITYGETGGNSPVQEFTVPKNVYTATISGLKPG extension (N + 8) and short tail)
VDYTITVYAVTRFRDYQPISINYRTEIDKGSGS fused via GS.sub.10 linker (GS10
is GSGSGSGSGSGSGSGSVSDVPRDLEVVAATPT SEQ ID NO: 11) to E1 (with
SLLISWVAGAEDYQYYRITYGETGGNSPVQEFT N-terminal extension (N + 8)
VPHDLVTATISGLKPGVDYTITVYAVTDMMHV and no tail) EYTEHPISINYRT 27
I1-GS10-E1: I/E tandem MGVSDVPRDLEVVAATPTSLLISWSARLKVAR having I1
(with N-terminal YYRITYGETGGNSPVQEFTVPKNVYTATISGLK extension (N +
10) and short PGVDYTITVYAVTRFRDYQPISINYRTEIDKGSG tail) fused via
GS.sub.10 linker SGSGSGSGSGSGSGSGSVSDVPRDLEVVAATP (GS10 is SEQ ID
NO: 11) to TSLLISWVAGAEDYQYYRITYGETGGNSPVQEF E1 (with N-terminal
extension TVPHDLVTATISGLKPGVDYTITVYAVTDMMH (N + 8) and Ser tail)
VEYTEHPISINYRTEIDKPSQ 28 I1-GS10-E1: I/E tandem
MGVSDVPRDLEVVAATPTSLLISWSARLKVAR having I1 (with N-terminal
YYRITYGETGGNSPVQEFTVPKNVYTATISGLK extension (N + 10) and short
PGVDYTITVYAVTRFRDYQPISINYRTEIDKGSG tail) fused via GS.sub.10 linker
SGSGSGSGSGSGSGSGSVSDVPRDLEVVAATP (GS10 is SEQ ID NO: 11) to
TSLLISWVAGAEDYQYYRITYGETGGNSPVQEF E1 (with N-terminal extension
TVPHDLVTATISGLKPGVDYTITVYAVTDMMH (N + 8) and Ser tail with his tag)
VEYTEHPISINYRTEIDKPSQHHHHHH 29 E1-GS10-I1: E/I tandem
VSDVPRDLEVVAATPTSLLISWVAGAEDYQYY having E1 (with N-terminal
RITYGETGGNSPVQEFTVPHDLVTATISGLKPG extension (N + 8) and short tail)
VDYTITVYAVTDMMHVEYTEHPISINYRTEIDK fused via GS.sub.10 linker (GS10
is GSGSGSGSGSGSGSGSGSGSVSDVPRDLEVVA SEQ ID NO: 11) to I1 (with N-
ATPTSLLISWSARLKVARYYRITYGETGGNSPV terminal extension (N + 8) and
QEFTVPKNVYTATISGLKPGVDYTITVYAVTRF no tail) RDYQPISINYRT 30
E1-GS10-I1: E/I tandem MGVSDVPRDLEVVAATPTSLLISWVAGAEDYQ having E1
(with N-terminal YYRITYGETGGNSPVQEFTVPHDLVTATISGLK extension (N +
10) and short PGVDYTITVYAVTDMMHVEYTEHPISINYRTEI tail) fused via
GS.sub.10 linker DKGSGSGSGSGSGSGSGSGSGSVSDVPRDLEV (GS10 is SEQ ID
NO: 11) to VAATPTSLLISWSARLKVARYYRITYGETGGNS I1 (with N-terminal
extension PVQEFTVPKNVYTATISGLKPGVDYTITVYAVT (N + 8) and Ser tail)
RFRDYQPISINYRTEIDKPSQ 31 E1-GS10-I1: E/I tandem
MGVSDVPRDLEVVAATPTSLLISWVAGAEDYQ having E1 (with N-terminal
YYRITYGETGGNSPVQEFTVPHDLVTATISGLK extension (N + 10) and short
PGVDYTITVYAVTDMMHVEYTEHPISINYRTEI tail) fused via GS.sub.10 linker
+E,DUS DK+EE GSGSGSGSGSGSGSGSGSGSVSDVPRDLEV (GS10 is SEQ ID NO: 11)
to VAATPTSLLISWSARLKVARYYRITYGETGGNS I1 (with N-terminal extension
PVQEFTVPKNVYTATISGLKPGVDYTITVYAVT (N + 8) and Ser tail with his
tag) RFRDYQPISINYRTEIDKPSQHHHHHH 32 .sup.10Fn3 scaffold, wherein
the VSDVPRDLEVVAATPTSLLI BC, DE, and FG loops are
(X).sub.nYYRITYGETGGNSPVQEFTV(X).sub.oATISGLKP represented by
(X).sub.n, (X).sub.o, and GVDYTITVYAV(X).sub.pISINYRT (X).sub.p,
respectively, and n is an integer from 1-20, o is an integer from
1-20, and p is an integer from 1-40 33 BC loop sequence from EGFR
SWVAGAEDYQ binder E1 34 BC loop sequence from EGFR
X.sub.mVAGAEDYQX.sub.n binder E1, wherein X is any amino acid and m
and n are independently selected from 0 to 5 amino acids 35 DE loop
sequence from EGFR PHDLVT binder E1 36 DE loop sequence from EGFR
X.sub.oHDLVX.sub.p binder E1, wherein X is any amino acid and o and
p are independently selected from 0 to 5 amino acids 37 FG loop
sequence from EGFR TDMMHVEYTEHP binder E1 38 FG loop sequence from
EGFR X.sub.qDMMHVEYTEHX.sub.r binder E1, wherein X is any amino
acid and q and r are independently selected from 0 to 5 amino acids
39 BC loop sequence from EGFR SWDSGRGSYQ binder E2 40 BC loop
sequence from EGFR X.sub.gDSGRGSYQX.sub.h binder E2, wherein X is
any amino acid and g and h are independently selected from 0 to 5
amino acids 41 DE loop sequence from EGFR PGPVHT binder E2 42 DE
loop sequence from EGFR X.sub.iGPVHX.sub.j binder E2, wherein X is
any amino acid and i and j are independently selected from 0 to 5
amino acids 43 FG loop sequence from EGFR TDHKPHADGPHTYHESP binder
E2 44 FG loop sequence from EGFR X.sub.kDHKPHADGPHTYHEX.sub.l
binder E2, wherein X is any amino acid and k and l are
independently selected from 0 to 5 amino acids 45 BC loop sequence
from SWSARLKVAR IGF-IR binder I1 46 BC loop sequence from
X.sub.aSARLKVAX.sub.b IGF-IR binder I1, wherein X is any amino acid
and a and b are independently selected from 0 to 5 amino acids 47
DE loop sequence from PKNVYT IGF-IR binder I1 48 DE loop sequence
from X.sub.cKNVYX.sub.d IGF-IR binder I1, wherein X is any amino
acid and c and d are independently selected from 0 to 5 amino acids
49 FG loop sequence from TRFRDYQP IGF-IR binder I1 50 FG loop
sequence from X.sub.eRFRDYQX.sub.f IGF-IR binder I1, wherein X is
any amino acid and e and f are independently selected from 0 to 5
amino acids
51 Linker GPG 52 E3 EGFR monomer with N-
MGVSDVPRDLEVVAATPTSLLISWLPGKLRYQ terminal extension (N + 10), Ser
YYRITYGETGGNSPVQEFTVPHDLRTATISGLK tail and his tag
PGVDYTITVYAVTNMMHVEYSEYPISINYRTEI DKPSQHHHHHH 53 E3-GS10-I1: E/I
tandem MGVSDVPRDLEVVAATPTSLLISWLPGKLRYQ having E3 (with N-terminal
YYRITYGETGGNSPVQEFTVPHDLRTATISGLK extension (N + 10) and short
PGVDYTITVYAVTNMMHVEYSEYPISINYRTEI tail) fused via GS.sub.10 linker
DKGSGSGSGSGSGSGSGSGSGSVSDVPRDLEV (GS10 is SEQ ID NO: 11) to
VAATPTSLLISWSARLKVARYYRITYGETGGNS I1 (with N-terminal extension
PVQEFTVPKNVYTATISGLKPGVDYTITVYAVT (N + 8) and Cys tail with his
RFRDYQPISINYRTEIDKPCQHHHHHH tag) 54 I1-GS10-E3: I/L tandem
MGVSDVPRDLEVVAATPTSLLISWSARLKVAR having I1 with N-terminal
YYRITYGETGGNSPVQEFTVPKNVYTATISGLK extension (N + 10) and short
PGVDYTITVYAVTRFRDYQPISINYRTEIDKGSG tail) fused via GS.sub.10 linker
SGSGSGSGSGSGSGSGSVSDVPRDLEVVAATP (GS10 is SEQ ID NO: 11) to
TSLLISWLPGKLRYQYYRITYGETGGNSPVQEF E3 (with N-terminal extension
TVPHDLRTATISGLKPGVDYTITVYAVTNMMH (N + 8) and Cys tail with his
VEYSEYPISINYRTEIDKPCQHHHHHH tag) 55 E1-GS10-I1: E/I tandem
MGVSDVPRDLEVVAATPTSLLISWVAGAEDYQ having E1 (with N-terminal
YYRITYGLTGGNSPVQLFTVPHDLVTATISGLK extension (N + 10) and short
PGVDYTITVYAVTDMMHVLYTLHPISINYRTEI tail) fused via GS.sub.10 linker
DKGSGSGSGSGSGSGSGSGSGSVSDVPRDLEV (GS10 is SEQ ID NO: 11) to
VAATPTSLLISWSARLKVARYYRITYGETGGNS I1 (with N-terminal extension
PVQEFTVPKNVYTATISGLKPGVDYTITVYAVT (N + 8) and Cys tail with his
RFRDYQPISINYRTEIDKPCQHHHHHH tag) 56 E2-GS10-I1: E/I tandem
MGVSDVPRDLEVVAATPTSLLISWDSGRGSYQ having E2 (with N-terminal
YYRITYGETGGNSPVQEFTVPGPVHTATISGLK extension (N + 10) and short
PGVDYTITVYAVTDHKPHADGPHTYHESPISIN tail) fused via GS.sub.10 linker
YRTEIDKGSGSGSGSGSGSGSGSGSGSVSDVP (GS10 is SEQ ID NO: 11) to
RDLEVVAATPTSLLISWSARLKVARYYRITYGE I1 (with N-terminal extension
TGGNSPVQEFTVPKNVYTATISGLKPGVDYTIT (N + 8) and Cys tail with his
VYAVTRFRDYQPISINYRTEIDKPCQHHHHHH tag) 57 I1-GS10-E1: I/E tandem
MGVSDVPRDLEVVAATPTSLLISWSARLKVAR having I1 (with N-terminal
YYRITYGETGGNSPVQEFTVPKNVYTATISGLK extension (N + 10) and short
PGVDYTITVYAVTRFRDYQPISINYRTEIDKGSG tail) fused via GS.sub.10 linker
SGSGSGSGSGSGSGSGSVSDVPRDLEVVAATP (GS10 is SEQ ID NO: 11) to
TSLLISWVAGAEDYQYYRITYGETGGNSPVQEF E1 (with N-terminal extension
TVPHDLVTATISGLKPGVDYTITVYAVTDMMH (N + 8) and Cys tail with his
VEYTEHPISINYRTEIDKPCQHHHHHH tag) 58 I1-GS10-E2: I/E tandem
MGVSDVPRDLEVVAATPTSLLISWSARLKVAR having I1 (with N-terminal
YYRITYGETGGNSPVQEFTVPKNVYTATISGLK extension (N + 10) and short
PGVDYTITVYAVTRFRDYQPISINYRTEIDKGSG tail) fused via GS.sub.10 linker
SGSGSGSGSGSGSGSGSVSDVPRDLEVVAATP (GS10 is SEQ ID NO: 11) to
TSLLISWDSGRGSYQYYRITYGETGGNSPVQEF E2 (with N-terminal extension
TVPGPVHTATISGLKPGVDYTITVYAVTDHKPH (N + 8) and Cys tail with his
ADGPHTYHESPISINYRTEIDKPCQHHHHHH tag) 59 BC loop sequence from EGFR
SWLPGKLRYQ binder E3 60 BC loop sequence from EGFR
X.sub.sLPGKLRYQX.sub.t binder E3, wherein X is any amino acid and s
and t are independently selected from 0 to 5 amino acids 61 DE loop
sequence from EGFR PHDLRT binder E3 62 DE loop sequence from EGFR
X.sub.uHDLRX.sub.w binder E3, wherein X is any amino acid and u and
w are independently selected from 0 to 5 amino acids 63 FG loop
sequence from EGFR TNMMHVEYSEYP binder E3 64 DE loop sequence from
EGFR X.sub.yNMMHVEYSEYX.sub.z binder E3, wherein X is any amino
acid and y and z are independently selected from 0 to 5 amino acids
65 I1 IGF-IR monomer core EVVAATPTSLLISWSARLKVARYYRITYGETGG
sequence: I1 without NSPVQEFTVPKNVYTATISGLKPGVDYTITVYA N-terminal
extension or C- VTRFRDYQPISINYRT terminal tail 66 E1 EGFR monomer
core EVVAATPTSLLISWVAGAEDYQYYRITYGETG sequence: E1 without
GNSPVQEFTVPHDLVTATISGLKPGVDYTITVY N-terminal extension or C-
AVTDMMHVEYTEHPISINYRT terminal tail 67 E2 EGFR monomer core
EVVAATPTSLLISWDSGRGSYQYYRITYGETGG sequence: E2 without
NSPVQEFTVPGPVHTATISGLKPGVDYTITVYA N-terminal extension or
VTDHKPHADGPHTYHESPISINYRT C-terminal tail 68 E3 EGFR monomer core
EVVAATPTSLLISWLPGKLRYQYYRITYGETGG sequence: SEQ ID NO: 82
NSPVQEFTVPHDLRTATISGLKPGVDYTITVYA without N-terminal extension
VTNMMHVEYSEYPISINYRT or C-terminal tail 69 Exemplary N-terminal
MGVSDVPRDL extension (N + 10) 70 Exemplary N-terminal GVSDVPRDL
extension 71 Exemplary N-terminal VSDVPRDL extension (N + 8) 72
Exemplary N-terminal X.sub.nSDVPRDL extension, wherein X is any
amino acid and n is 0, 1 or 2, preferably when n = 1, X is Met or
Gly and when n = 2, X is Met-Gly 73 Exemplary N-terminal
X.sub.nDVPRDL extension, wherein X is any amino acid and n is 0, 1
or 2, preferably when n = 1, X is Met or Gly and when n = 2, X is
Met-Gly 74 Exemplary N-terminal X.sub.nVPRDL extension, wherein X
is any amino acid and n is 0, 1 or 2, preferably when n = 1, X is
Met or Gly and when n = 2, X is Met-Gly 75 Exemplary N-terminal
X.sub.nPRDL extension, wherein X is any amino acid and n is 0, 1 or
2, preferably when n = 1, X is Met or Gly and when n = 2, X is
Met-Gly 76 Exemplary N-terminal X.sub.nRDL extension, wherein X is
any amino acid and n is 0, 1 or 2, preferably when n = 1, X is Met
or Gly and when n = 2, X is Met-Gly 77 Exemplary N-terminal
X.sub.nDL extension, wherein X is any amino acid and n is 0, 1 or
2, preferably when n = 1, X is Met or Gly and when n = 2, X is
Met-Gly 78 Short tail EIDK 79 Exemplary C-terminal tail EIDKP 80
Exemplary C-terminal tail EIDKPS 81 Exemplary C-terminal tail
EIDKPC 82 E3 EGFR monomer with N- VSDVPRDLEVVAATPTSLLISWLPGKLRYQYY
terminal extension (N + 8) and RITYGETGGNSPVQEFTVPHDLRTATISGLKPG no
tail VDYTITVYAVTNMMHVEYSEYPISINYRT 87 E3-GS10-I1: E/I tandem
VSDVPRDLEVVAATPTSLLISWLPGKLRYQYY having E3 (with N-terminal
RITYGETGGNSPVQEFTVPHDLRTATISGLKPG extension (N + 8) and short tail)
VDYTITVYAVTNMMHVEYSEYPISINYRTEIDK fused via GS.sub.10 linker (GS10
is GSGSGSGSGSGSGSGSGSGSVSDVPRDLEVVA SEQ ID NO: 11) to I1 (with N-
ATPTSLLISWSARLKVARYYRITYGETGGNSPV terminal extension (N + 8) and
QEFTVPKNVYTATISGLKPGVDYTITVYAVTRF Cys tail) RDYQPISINYRTEIDKPCQ 88
I1-GS10-E3: I/E tandem VSDVPRDLEVVAATPTSLLISWSARLKVARYY having I1
(with N-terminal RITYGETGGNSPVQEFTVPKNVYTATISGLKPG extension (N +
8) and short tail) VDYTITVYAVTRFRDYQPISINYRTEIDKGSGS fused via
GS.sub.10 linker (GS10 is GSGSGSGSGSGSGSGSVSDVPRDLEVVAATPT SEQ ID
NO: 11) to E3 (with SLLISWLPGKLRYQYYRITYGETGGNSPVQEFT N-terminal
extension (N + 8) VPHDLRTATISGLKPGVDYTITVYAVTNMMHV and Cys tail)
EYSEYPISINYRTEIDKPCQ 89 E1-GS10-I1: E/I tandem
VSDVPRDLEVVAATPTSLLISWVAGAEDYQYY having E1 (with N-terminal
RITYGETGGNSPVQEFTVPHDLVTATISGLKPG extension (N + 8) and short tail)
VDYTITVYAVTDMMHVEYTEHPISINYRTEIDK fused via GS.sub.10 linker (GS10
is GSGSGSGSGSGSGSGSGSGSVSDVPRDLEVVA SEQ ID NO: 11) to I1 (with N-
ATPTSLLISWSARLKVARYYRITYGETGGNSPV terminal extension (N + 8) and
QEFTVPKNVYTATISGLKPGVDYTITVYAVTRF Cys tail) RDYQPISINYRTEIDKPCQ 90
E2-GS10-I1: E/I tandem VSDVPRDLEVVAATPTSLLISWDSGRGSYQYY having E2
(with N-terminal RITYGETGGNSPVQEFTVPGPVHTATISGLKPG extension (N +
8) and short tail) VDYTITVYAVTDHKPHADGPHTYHESPISINYR fused via
GS.sub.10 linker (GS10 is TEIDKGSGSGSGSGSGSGSGSGSGSVSDVPRD SEQ ID
NO: 11) to I1 (with N- LEVVAATPTSLLISWSARLKVARYYRITYGETG terminal
extension (N + 8) and GNSPVQEFTVPKNVYTATISGLKPGVDYTITVY Cys tail)
AVTRFRDYQPISINYRTEIDKPCQ 91 I1-GS10-E1: I/E tandem
VSDVPRDLEVVAATPTSLLISWSARLKVARYY having I1 (with N-terminal
RITYGETGGNSPVQEFTVPKNVYTATISGLKPG extension (N + 8) and short tail)
VDYTITVYAVTRFRDYQPISINYRTEIDKGSGS fused via GS.sub.10 linker (GS10
is GSGSGSGSGSGSGSGSVSDVPRDLEVVAATPT SEQ ID NO: 11) to E1 (with
SLLISWVAGAEDYQYYRITYGETGGNSPVQEFT N-terminal extension (N + 8)
VPHDLVTATISGLKPGVDYTITVYAVTDMMHV and Cys tail) EYTEHPISINYRTEIDKPCQ
92 I1-GS10-E2: I/E tandem VSDVPRDLEVVAATPTSLLISWSARLKVARYY having
Ii (with N-terminal RITYGETGGNSPVQEFTVPKNVYTATISGLKPG extension (N
+ 8) and short tail) VDYTITVYAVTRFRDYQPISINYRTEIDKGSGS fused via
GS.sub.10 linker (GS10 is GSGSGSGSGSGSGSGSVSDVPRDLEVVAATPT SEQ ID
NO: 11) to E2 (with SLLISWDSGRGSYQYYRITYGETGGNSPVQEFT N-terminal
extension (N + 8) VPGPVHTATISGLKPGVDYTITVYAVTDHKPHA and Cys tail)
DGPHTYHESPISINYRTEIDKPCQ 93 PA3 Linker PAPAPA 94 PA6 Linker
PAPAPAPAPAPA 95 PA9 Linker PAPAPAPAPAPAPAPAPA 96 Modified Ser tail
EGSGS 97 Modified Cys tail EGSGC 98 E3-(PA).sub.n-I1: E/I tandem
VSDVPRDLEVVAATPTSLLISWLPGKLRYQYY having E3 (with N-terminal
RITYGETGGNSPVQEFTVPHDLRTATISGLKPG extension (N + 8) and an E tail)
VDYTITVYAVTNMMHVEYSEYPISINYRTE(PA).sub.n fused via (PA).sub.n
linker ((PA).sub.n is VSDVPRDLEVVAATPTSLLISWSARLKVARYY SEQ ID NO:
488) to I1 (with RITYGETGGNSPVQEFTVPKNVYTATISGLKPG N-terminal
extension (N + 8) VDYTITVYAVTRFRDYQPISINYRTEGSGX and a modified Ser
or Cys tail), wherein n = 3, 6 or 9, and X = Ser or Cys 99
I1-(PA).sub.n-E3: I/E tandem VSDVPRDLEVVAATPTSLLISWSARLKVARYY
having I1 (with N-terminal RITYGETGGNSPVQEFTVPKNVYTATISGLKPG
extension (N + 8) and an E tail)
VDYTITVYAVTRFRDYQPISINYRTE(PA).sub.nVSDV fused via (PA).sub.n
linker ((PA).sub.n is PRDLEVVAATPTSLLISWLPGKLRYQYYRITYG SEQ ID NO:
488) to E3 (with ETGGNSPVQEFTVPHDLRTATISGLKPGVDYTI N-terminal
extension (N + 8) TVYAVTNMMHVEYSEYPISINYRTEGSGX and a modified Ser
or Cys tail),
wherein n = 3, 6 or 9, and X = Ser or Cys 100 E1-(PA).sub.n-I1: E/I
tandem VSDVPRDLEVVAATPTSLLISWVAGAEDYQYY having E1 (with N-terminal
RITYGETGGNSPVQEFTVPHDLVTATISGLKPG extension (N + 8) and an E tail)
VDYTITVYAVTDMMHVEYTEHPISINYRTE(PA).sub.n fused via (PA).sub.n
linker ((PA).sub.n is VSDVPRDLEVVAATPTSLLISWSARLKVARYY SEQ ID NO:
488) to I1 (with RITYGETGGNSPVQEFTVPKNVYTATISGLKPG N-terminal
extension (N + 8) VDYTITVYAVTRFRDYQPISINYRTEGSGX and a modified Ser
or Cys tail), wherein n = 3, 6 or 9, and X = Ser or Cys 101
E2-(PA).sub.n-I1: E/I tandem VSDVPRDLEVVAATPTSLLISWDSGRGSYQYY
having E2 (with N-terminal RITYGETGGNSPVQEFTVPGPVHTATISGLKPG
extension (N + 8) and an E tail) VDYTITVYAVTDHKPHADGPHTYHESPISINYR
fused via (PA).sub.n linker ((PA).sub.n is
TE(PA).sub.nVSDVPRDLEVVAATPTSLLISWSARLK SEQ ID NO: 488) to I1 (with
VARYYRITYGETGGNSPVQEFTVPKNVYTATIS N-terminal extension (N + 8)
GLKPGVDYTITVYAVTRFRDYQPISINYRTEGSGX and a modified Ser or Cys
tail), wherein n = 3, 6 or 9, and X = Ser or Cys 102
I1-(PA).sub.n-E1: I/E tandem VSDVPRDLEVVAATPTSLLISWSARLKVARYY
having I1 (with N-terminal RITYGETGGNSPVQEFTVPKNVYTATISGLKPG
extension (N + 8) and an E tail)
VDYTITVYAVTRFRDYQPISINYRTE(PA).sub.nVSDV fused via (PA).sub.n
linker ((PA).sub.n is PRDLEVVAATPTSLLISWVAGAEDYQYYRITY SEQ ID NO:
488) to E1 (with GETGGNSPVQEFTVPHDLVTATISGLKPGVDYT N-terminal
extension (N + 8) ITVYAVTDMMHVEYTEHPISINYRTEGSGX and a modified Ser
or Cys tail), wherein n = 3, 6 or 9, and X = Ser or Cys 103
I1-(PA).sub.n-E2: I/E tandem VSDVPRDLEVVAATPTSLLISWSARLKVARYY
having I1 (with N-terminal RITYGETGGNSPVQEFTVPKNVYTATISGLKPG
extension (N + 8) and an E tail)
VDYTITVYAVTRFRDYQPISINYRTE(PA)nVSDV fused via (PA).sub.n linker
((PA).sub.n is PRDLEVVAATPTSLLISWDSGRGSYQYYRITYG SEQ ID NO: 488) to
E2 (with ETGGNSPVQEFTVPGPVHTATISGLKPGVDYTI N-terminal extension (N
+ 8) TVYAVTDHKPHADGPHTYHESPISINYRTGSGX and a modified Ser or Cys
tail), wherein n = 3, 6 or 9, and X = Ser or Cys 104 E3-GS10-I1:
E/I tandem VSDVPRDLEVVAATPTSLLISWLPGKLRYQYY having E3 (with
N-terminal RITYGETGGNSPVQEFTVPHDLRTATISGLKPG extension (N + 8) and
short tail) VDYTITVYAVTNMMHVEYSEYPISINYRTEIDK fused via GS.sub.10
linker (GS10 is GSGSGSGSGSGSGSGSGSGSVSDVPRDLEVVA SEQ ID NO: 11) to
I1 (with N- ATPTSLLISWSARLKVARYYRITYGETGGNSPV terminal extension (N
+ 8) and QEFTVPKNVYTATISGLKPGVDYTITVYAVTRF Ser tail)
RDYQPISINYRTEIDKPSQ 105 I1-GS10-E3: I/E tandem
VSDVPRDLEVVAATPTSLLISWSARLKVARYY having I1 with N-terminal
RITYGETGGNSPVQEFTVPKNVYTATISGLKPG extension (N + 8) and short tail)
VDYTITVYAVTRFRDYQPISINYRTEIDKGSGS fused via GS.sub.10 linker (GS10
is GSGSGSGSGSGSGSGSVSDVPRDLEVVAATPT SEQ ID NO: 11) to E3 (with
SLLISWLPGKLRYQYYRITYGETGGNSPVQEFT N-terminal extension (N + 8)
VPHDLRTATISGLKPGVDYTITVYAVTNMMHV and Ser tail) EYSEYPISINYRTEIDKPSQ
106 E4 EGFR monomer with N- VSDVPRDLEVVAATPTSLLISWHERDGSRQYY
terminal extension (N + 8) and RITYGETGGNSPVQEFTVPGGVRTATISGLKPG no
tail VDYTITVYAVTDYFNPTTHEYIYQTTPISINYRT 107 E4 EGFR monomer with N-
MGVSDVPRDLEVVAATPTSLLISWHERDGSRQ terminal extension (N + 10) and
YYRITYGETGGNSPVQEFTVPGGVRTATISGLK a Ser with His tag
PGVDYTITVYAVTDYFNPTTHEYIYQTTPISINY RTEIDKPSQHHHHHH 108 E4 EGFR
monomer core EVVAATPTSLLISWHERDGSRQYYRITYGETGG sequence: E4 without
N- NSPVQEFTVPGGVRTATISGLKPGVDYTITVYA terminal extension or C-
VTDYFNPTTHEYIYQTTPISINYRT terminal tail 109 BC loop sequence from
EGFR SWHERDGSRQ binder E4 110 DE loop sequence from EGFR PGGVRT
binder E4 111 FG loop sequence from EGFR TDYFNPTTHEYIYQTTP binder
E4 112 E5 EGFR monomer with N- VSDVPRDLEVVAATPTSLLISWWAPVDRYQYY
terminal extension (N + 8) and RITYGETGGNSPVQEFTVPRDVYTATISGLKPG no
tail VDYTITVYAVTDYKPHADGPHTYHESPISINYR T 113 E5 EGFR monomer with
N- MGVSDVPRDLEVVAATPTSLLISWWAPVDRYQ terminal extension (N + 10) and
YYRITYGETGGNSPVQEFTVPRDVYTATISGLK a modified Ser or Cys tail,
PGVDYTITVYAVTDYKPHADGPHTYHESPISIN wherein X = Ser or Cys; may
YRTEIDKPXQ optionally comprise a 6X His tag (SEQ ID NO: 487) 114 ES
EGFR monomer core EVVAATPTSLLISWWAPVDRYQYYRITYGETG sequence: ES
without N- GNSPVQEFTVPRDVYTATISGLKPGVDYTITVY terminal extension or
C- AVTDYKPHADGPHTYHESPISINYRT terminal tail 115 BC loop sequence
from EGFR SWWAPVDRYQ binder E5 116 DE loop sequence from EGFR
PRDVYT binder E5 117 FG loop sequence from EGFR TDYKPHADGPHTYHESP
binder E5 118 E4-GS10-I1: E/I tandem
VSDVPRDLEVVAATPTSLLISWHERDGSRQYY having E4 (with N-terminal
RITYGETGGNSPVQEFTVPGGVRTATISGLKPG extension (N + 8) and short tail)
VDYTITVYAVTDYFNPTTHEYIYQTTPISINYRT fused via GS10 linker (GS10 is
EIDKGSGSGSGSGSGSGSGSGSGSVSDVPRDL SEQ ID NO: 11) to I1 (with N-
EVVAATPTSLLISWSARLKVARYYRITYGETGG terminal extension (N + 8) and
NSPVQEFTVPKNVYTATISGLKPGVDYTITVYA no tail) VTRFRDYQPISINYRT 119
E4-GS10-I1: E/I tandem VSDVPRDLEVVAATPTSLLISWHERDGSRQYY having E4
(with N-terminal RITYGETGGNSPVQEFTVPGGVRTATISGLKPG extension (N +
8) and short tail) VDYTITVYAVTDYFNPTTHEYIYQTTPISINYRT fused via
GS10 linker (GS10 is EIDKGSGSGSGSGSGSGSGSGSGSVSDVPRDL SEQ ID NO:
11) to I1 (with N- EVVAATPTSLLISWSARLKVARYYRITYGETGG terminal
extension (N + 8) and NSPVQEFTVPKNVYTATISGLKPGVDYTITVYA modified
Ser or Cys tail), VTRFRDYQPISINYRTEIDKPXQ wherein X = Ser or Cys;
may optionally comprise a 6X His tag (SEQ ID NO: 487) 120
E4-GS10-I1: E/I tandem MGVSDVPRDLEVVAATPTSLLISWHERDGSRQ having E4
(with N-terminal YYRITYGETGGNSPVQEFTVPGGVRTATISGLK extension (N +
10) and short PGVDYTITVYAVTDYFNPTTHEYIYQTTPISINY tail) fused via
GS10 linker RTEIDKGSGSGSGSGSGSGSGSGSGSVSDVPR (GS10 is SEQ ID NO:
11) to DLEVVAATPTSLLISWSARLKVARYYRITYGET I1 (with N-terminal
extension GGNSPVQEFTVPKNVYTATISGLKPGVDYTITV (N + 8) and Cys tail)
with his YAVTRFRDYQPISINYRTEIDKPCQHHHHHH tag 121 E4-(PA).sub.n-I1:
E/I tandem VSDVPRDLEVVAATPTSLLISWHERDGSRQYY having E4 (with
N-terminal RITYGETGGNSPVQEFTVPGGVRTATISGLKPG extension (N + 8) and
an E tail) VDYTITVYAVTDYFNPTTHEYIYQTTPISINYRT fused via (PA).sub.n
linker ((PA).sub.n is E(PA).sub.nVSDVPRDLEVVAATPTSLLISWSARLKV SEQ
ID NO: 488) to I1 (with ARYYRITYGETGGNSPVQEFTVPKNVYTATISG
N-terminal extension (N + 8) LKPGVDYTITVYAVTRFRDYQPISINYRTEIDKP and
modified Ser or Cys tail), CQHHHHHH wherein n = 3, 6 or 9, and X =
Ser or Cys; may optionally comprise a 6X His tag (SEQ ID NO: 487)
122 I1-GS10-E4: I/E tandem VSDVPRDLEVVAATPTSLLISWSARLKVARYY having
I1 (with N-terminal RITYGETGGNSPVQEFTVPKNVYTATISGLKPG extension (N
+ 8) and short tail) VDYTITVYAVTRFRDYQPISINYRTEIDKGSGS fused via
GS10 linker (GS10 is GSGSGSGSGSGSGSGSVSDVPRDLEVVAATPT SEQ ID NO:
11) to E4 (having SLLISWHERDGSRQYYRITYGETGGNSPVQEFT N-terminal
extension (N + 8) VPGGVRTATISGLKPGVDYTITVYAVTDYFNPT and no tail)
THEYIYQTTPISINYRT 123 I1-GS10-E4: I/E tandem
VSDVPRDLEVVAATPTSLLISWSARLKVARYY having I1 (with N-terminal
RITYGETGGNSPVQEFTVPKNVYTATISGLKPG extension (N + 8) and short tail)
VDYTITVYAVTRFRDYQPISINYRTEIDKGSGS fused via GS10 linker (GS10 is
GSGSGSGSGSGSGSGSVSDVPRDLEVVAATPT SEQ ID NO: 11) to E4 (with
SLLISWHERDGSRQYYRITYGETGGNSPVQEFT N-terminal extension (N + 8)
VPGGVRTATISGLKPGVDYTITVYAVTDYFNPT and modified Ser or Cys tail),
THEYIYQTTPISINYRTEIDKPXQ wherein X = Ser or Cys; may optionally
comprise a 6X His tag (SEQ ID NO: 487) 124 I1-GS10-E4: I/E tandem
MGVSDVPRDLEVVAATPTSLLISWSARLKVAR having I1 (with N-terminal
YYRITYGETGGNSPVQEFTVPKNVYTATISGLK extension (N + 10) and short
PGVDYTITVYAVTRFRDYQPISINYRTEIDKGSG tail) fused via GS10 linker
SGSGSGSGSGSGSGSGSVSDVPRDLEVVAATP (GS10 is SEQ ID NO: 11) to
TSLLISWHERDGSRQYYRITYGETGGNSPVQEF E4 (with N-terminal extension
TVPGGVRTATISGLKPGVDYTITVYAVTDYFNP (N + 8) and a Cys tail) with his
TTHEYIYQTTPISINYRTEIDKPCQHHHHHH tag 125 I1-(PA).sub.n-E4: I/E
tandem VSDVPRDLEVVAATPTSLLISWSARLKVARYY having I1 (with N-terminal
RITYGETGGNSPVQEFTVPKNVYTATISGLKPG extension (N + 8) and an E tail)
VDYTITVYAVTRFRDYQPISINYRTE(PA).sub.nVSDV fused via (PA).sub.n
linker ((PA).sub.n is PRDLEVVAATPTSLLISWHERDGSRQYYRITYG SEQ ID NO:
488) to E4 (with ETGGNSPVQEFTVPGGVRTATISGLKPGVDYTI N-terminal
extension (N + 8) TVYAVTDYFNPTTHEYIYQTTPISINYRTEIDKP and modified
Ser or Cys tail), XQ wherein n = 3, 6 or 9, and X = Ser or Cys; may
optionally comprise a 6X His tag (SEQ ID NO: 487) 126 E5-GS10-I1:
E/I tandem VSDVPRDLEVVAATPTSLLISWWAPVDRYQYY having E5 (with
N-terminal RITYGETGGNSPVQEFTVPRDVYTATISGLKPG extension (N + 8) and
short tail) VDYTITVYAVTDYKPHADGPHTYHESPISINYR fused via GS10 linker
(GS10 is TEIDKGSGSGSGSGSGSGSGSGSGSVSDVPRD SEQ ID NO: 11) to I1
(with N- LEVVAATPTSLLISWSARLKVARYYRITYGETG terminal extension (N +
8) and GNSPVQEFTVPKNVYTATISGLKPGVDYTITVY no tail) AVTRFRDYQPISINYRT
127 E5-GS10-I1: E/I tandem VSDVPRDLEVVAATPTSLLISWWAPVDRYQYY having
E5 (with N-terminal RITYGETGGNSPVQEFTVPRDVYTATISGLKPG extension (N
+ 8) and short tail) VDYTITVYAVTDYKPHADGPHTYHESPISINYR fused via
GS10 linker (GS10 is TEIDKGSGSGSGSGSGSGSGSGSGSVSDVPRD SEQ ID NO:
11) to I1 (with N- LEVVAATPTSLLISWSARLKVARYYRITYGETG terminal
extension (N + 8) and GNSPVQEFTVPKNVYTATISGLKPGVDYTITVY modified
Ser or Cys tail), AVTRFRDYQPISINYRTEIDKPXQ wherein X = Ser or Cys;
may optionally comprise a 6X His tag (SEQ ID NO: 487) 128
E5-GS10-I1: E/I tandem MGVSDVPRDLEVVAATPTSLLISWWAPVDRYQ having E5
(with N-terminal YYRITYGETGGNSPVQEFTVPRDVYTATISGLK extension (N +
10) and short PGVDYTITVYAVTDYKPHADGPHTYHESPISIN tail) fused via
GS10 linker YRTEIDKGSGSGSGSGSGSGSGSGSGSVSDVP (GS10 is SEQ ID NO:
11) to RDLEVVAATPTSLLISWSARLKVARYYRITYGE I1 (with N-terminal
extension TGGNSPVQEFTVPKNVYTATISGLKPGVDYTIT (N + 8) and a Cys
tail), with a VYAVTRFRDYQPISINYRTEIDKPCQHHHHHH His tag 129
E5-(PA).sub.n-I1: E/I tandem VSDVPRDLEVVAATPTSLLISWWAPVDRYQYY
having E5 (with N-terminal RITYGETGGNSPVQEFTVPRDVYTATISGLKPG
extension (N + 8) and an E tail) VDYTITVYAVTDYKPHADGPHTYHESPISINYR
fused via (PA).sub.n linker ((PA).sub.n is
TE(PA).sub.nVSDVPRDLEVVAATPTSLLISWSARLK SEQ ID NO: 488) to I1 (with
VARYYRITYGETGGNSPVQEFTVPKNVYTATIS N-terminal extension (N + 8)
GLKPGVDYTITVYAVTRFRDYQPISINYRTEIDK and modified Ser or Cys tail),
PXQ wherein n = 3, 6 or 9, and X = Ser or Cys; may optionally
comprise a 6X His tag (SEQ ID NO: 487) 130 I1-GS10-E5: I/E tandem
VSDVPRDLEVVAATPTSLLISWSARLKVARYY having I1 (with N-terminal
RITYGETGGNSPVQEFTVPKNVYTATISGLKPG extension (N + 8) and short tail)
VDYTITVYAVTRFRDYQPISINYRTEIDKGSGS fused via GS10 linker (GS10 is
GSGSGSGSGSGSGSGSVSDVPRDLEVVAATPT SEQ ID NO: 11) to E5 (with
SLLISWWAPVDRYQYYRITYGETGGNSPVQEF N-terminal extension (N + 8)
TVPRDVYTATISGLKPGVDYTITVYAVTDYKPH and no tail) ADGPHTYHESPISINYRT
131 I1-GS10-E5: I/E tandem VSDVPRDLEVVAATPTSLLISWSARLKVARYY having
I1 (with N-terminal RITYGETGGNSPVQEFTVPKNVYTATISGLKPG extension (N
+ 8) and short tail) VDYTITVYAVTRFRDYQPISINYRTEIDKGSGS fused via
GS10 linker (GS10 is GSGSGSGSGSGSGSGSVSDVPRDLEVVAATPT SEQ ID NO:
11) to E5 (with SLLISWWAPVDRYQYYRITYGETGGNSPVQEF
N-terminal extension (N + 8) TVPRDVYTATISGLKPGVDYTITVYAVTDYKPH and
modified Ser or Cys tail), ADGPHTYHESPISINYRTEIDKPXQ wherein X =
Ser or Cys; may optionally comprise a 6X His tag (SEQ ID NO: 487)
132 I1-GS10-E5: I/E tandem MGVSDVPRDLEVVAATPTSLLISWSARLKVAR having
I1 (with N-terminal YYRITYGETGGNSPVQEFTVPKNVYTATISGLK extension (N
+ 10) and short PGVDYTITVYAVTRFRDYQPISINYRTEIDKGSG tail) fused via
GS10 linker SGSGSGSGSGSGSGSGSVSDVPRDLEVVAATP (GS10 is SEQ ID NO:
11) to TSLLISWWAPVDRYQYYRITYGETGGNSPVQE E5 (with N-terminal
extension FTVPRDVYTATISGLKPGVDYTITVYAVTDYKP (N + 8) and a Cys tail)
with a HADGPHTYHESPISINYRTEIDKPCQHHHHHH His tag 133
I1-(PA).sub.n-E5: I/E tandem MGVSDVPRDLEVVAATPTSLLISWSARLKVAR
having I1 (with N-terminal YYRITYGETGGNSPVQEFTVPKNVYTATISGLK
extension (N + 8) and an E tail)
PGVDYTITVYAVTRFRDYQPISINYRTE(PA).sub.nVS fused via (PA).sub.n
linker ((PA).sub.n is DVPRDLEVVAATPTSLLISWWAPVDRYQYYRI SEQ ID NO:
488) to E5 (with TYGETGGNSPVQEFTVPRDVYTATISGLKPGVD N-terminal
extension (N + 8) YTITVYAVTDYKPHADGPHTYHESPISINYRTEI and modified
Ser or Cys tail), DKPXQ wherein n = 3, 6 or 9, and X = Ser or Cys;
may optionally comprise a 6X His tag (SEQ ID NO: 487) 134 BC loop
sequence from EGFR X.sub.gHERDGSRQX.sub.h binder E4, wherein X is
any amino acid and g and h are independently selected from 0 to 5
amino acids 135 DE loop sequence from EGFR X.sub.iGGVRX.sub.j
binder E4, wherein X is any amino acid and i and j are
independently selected from 0 to 5 amino acids 136 FG loop sequence
from EGFR X.sub.kDYFNPTTHEYIYQTTX.sub.l binder E4, wherein X is any
amino acid and k and l are independently selected from 0 to 5 amino
acids 137 BC loop sequence from EGFR X.sub.gWAPVDRYQX.sub.h binder
E5, wherein X is any amino acid and g and h are independently
selected from 0 to 5 amino acids 138 DE loop sequence from EGFR
X.sub.iRDVYX.sub.j binder E5, wherein X is any amino acid and i and
j are independently selected from 0 to 5 amino acids 139 FG loop
sequence from EGFR X.sub.kDYKPHADGPHTYHESX.sub.l binder E5, wherein
X is any amino acid and k and l are independently selected from 0
to 5 amino acids 140 E85 EGFR monomer with N-
MGVSDVPRDLEVVAATPTSLLISWTQGSTHYQ terminal extension (N + 10) and
YYRITYGETGGNSPVQEFTVPGMVYTATISGLK Ser tail with his tag
PGVDYTITVYAVTDYFDRSTHEYKYRTTPISIN YRTEIDKPSQHHHHHH 141 E85 EGFR
monomer core: EVVAATPTSLLISWTQGSTHYQYYRITYGETGG E85 monomer without
N- NSPVQEFTVPGMVYTATISGLKPGVDYTITVYA terminal extension or C-
VTDYFDRSTHEYKYRTTPISINYRT terminal tail 142 E85 EGFR monomer,
wherein X.sub.1EVVAATPTSLLISWTQGSTHYQYYRITYGET X.sub.1 is selected
from the group GGNSPVQEFTVPGMVYTATISGLKPGVDYTITV consisting of SEQ
ID NOs: 69- YAVTDYFDRSTHEYKYRTTPISINYRTX.sub.2 77 and X.sub.2 is
selected from the group consisting of SEQ ID NOs: 9, 10, or 78-81;
in exemplary emobidments, X.sub.1 is SEQ ID NO: 69 or 71 and
X.sub.2 is SEQ ID NO: 9 or 10; may optionally comprise a his tag
143 BC loop sequence from EGFR SWTQGSTHYQ binder E85 144 DE loop
sequence from EGFR PGMVYT binder E85 145 FG loop sequence from EGFR
TDYFDRSTHEYKYRTTP binder E85 146 BC loop sequence from EGFR
X.sub.gTQGSTHYQX.sub.h binder E85, wherein X is any amino acid and
g and h are independently selected from 0 to 5 amino acids 147 DE
loop sequence from EGFR X.sub.iGMVYX.sub.j binder E85, wherein X is
any amino acid and i and j are independently selected from 0 to 5
amino acids 148 FG loop sequence from EGFR
X.sub.kDYFDRSTHEYKYRTTX.sub.l binder E85, wherein X is any amino
acid and k and l are independently selected from 0 to 5 amino acids
149 E85-GS10-I1: E/I tandem MGVSDVPRDLEVVAATPTSLLISWTQGSTHYQ having
E85 (with N-terminal YYRITYGETGGNSPVQEFTVPGMVYTATISGLK extension (N
+ 10) and a short PGVDYTITVYAVTDYFDRSTHEYKYRTTPISIN tail) fused via
GS10 linker YRTEIDKGSGSGSGSGSGSGSGSGSGSVSDVP (GS10 is SEQ ID NO:
11) to RDLEVVAATPTSLLISWSARLKVARYYRITYGE I1 (with N-terminal
extension TGGNSPVQEFTVPKNVYTATISGLKPGVDYTIT (N + 8) and Cys tail)
with a 6X VYAVTRFRDYQPISINYRTEIDKPCQHHHHHH His tag (SEQ ID NO: 487)
150 E85-GS10-I1 core, wherein X.sub.1
X.sub.1EVVAATPTSLLISWTQGSTHYQYYRITYGET is optional and when present
is GGNSPVQEFTVPGMVYTATISGLKPGVDYTITV selected from the group
YAVTDYFDRSTHEYKYRTTPISINYRTEIDKGS consisting of SEQ ID NOs: 69-
GSGSGSGSGSGSGSGSGSVSDVPRDLEVVAAT 77, X.sub.2 is optional and when
PTSLLISWSARLKVARYYRITYGETGGNSPVQE present is selected from the
FTVPKNVYTATISGLKPGVDYTITVYAVTRFRD group consisting of SEQ ID
YQPISINYRTX.sub.2 NOs: 9, 10, or 78-81, and n = 3, 6 or 9; in
exemplary embodiments, X1 is SEQ ID NO: 69 or 71 and X.sub.2 is SEQ
ID NO: 9 or 10 151 E85-(PA).sub.n-I1 core, wherein X.sub.1
X.sub.1EVVAATPTSLLISWTQGSTHYQYYRITYGET is optional and when present
is GGNSPVQEFTVPGMVYTATISGLKPGVDYTITV selected from the group
YAVTDYFDRSTHEYKYRTTPISINYRTE(PA).sub.nVS consisting of SEQ ID NOs:
69- DVPRDLEVVAATPTSLLISWSARLKVARYYRIT 77, X.sub.2 is optional and
when YGETGGNSPVQEFTVPKNVYTATISGLKPGVD present is selected from the
YTITVYAVTRFRDYQPISINYRTX.sub.2 group consisting of SEQ ID NOs: 9,
10, or 78-81, and n = 3, 6 or 9; in exemplary embodiments, X.sub.1
is SEQ ID NO: 69 or 71 and X.sub.2 is SEQ ID NO: 9 or 10 152
I1-GS10-E85: I/E tandem MGVSDVPRDLEVVAATPTSLLISWSARLKVAR having I1
(with N-terminal YYRITYGETGGNSPVQEFTVPKNVYTATISGLK extension (N +
10) and a short PGVDYTITVYAVTRFRDYQPISINYRTEIDKGSG tail) fused via
GS10 linker SGSGSGSGSGSGSGSGSVSDVPRDLEVVAATP (GS10 is SEQ ID NO:
11) to TSLLISWTQGSTHYQYYRITYGETGGNSPVQEF E85 (with N-terminal
TVPGMVYTATISGLKPGVDYTITVYAVTDYFD extension (N + 8) and Cys tail)
RSTHEYKYRTTPISINYRTEIDKPCQHHHHHH with a 6X His tag (SEQ ID NO: 487)
153 I1-GS10-E85 core, wherein X.sub.1
X.sub.1EVVAATPTSLLISWSARLKVARYYRITYGET is optional and when present
is GGNSPVQEFTVPKNVYTATISGLKPGVDYTITV selected from the group
YAVTRFRDYQPISINYRTEIDKGSGSGSGSGSG consisting of SEQ ID NOs: 69-
SGSGSGSGSVSDVPRDLEVVAATPTSLLISWTQ 77, X.sub.2 is optional and when
GSTHYQYYRITYGETGGNSPVQEFTVPGMVYT present is selected from the
ATISGLKPGVDYTITVYAVTDYFDRSTHEYKYR group consisting of SEQ ID
TTPISINYRTX.sub.2 NOs: 9, 10, or 78-81, and n = 3, 6 or 9; in
exemplary embodiments, X.sub.1 is SEQ ID NO: 69 or 71 and X.sub.2
is SEQ ID NO: 9 or 10 154 I1-(PA).sub.n-E85 core, wherein X.sub.1
X.sub.1EVVAATPTSLLISWSARLKVARYYRITYGET is optional and when present
is GGNSPVQEFTVPKNVYTATISGLKPGVDYTITV selected from the group
YAVTRFRDYQPISINYRTE(PA).sub.nVSDVPRDLEV consisting of SEQ ID NOs:
69- VAATPTSLLISWTQGSTHYQYYRITYGETGGNS 77, X.sub.2 is optional and
when PVQEFTVPGMVYTATISGLKPGVDYTITVYAVT present is selected from the
DYFDRSTHEYKYRTTPISINYRTX.sub.2 group consisting of SEQ ID NOs: 9,
10, or 78-81, and n = 3, 6 or 9; in exemplary embodiments, X.sub.1
is SEQ ID NO: 69 or 71 and X.sub.2 is SEQ ID NO: 9 or 10 155 E90
EGFR monomer with N- MGVSDVPRDLEVVAATPTSLLISWYWEGLPYQ terminal
extension (N + 10) and YYRITYGETGGNSPVQEFTVPRDVNTATISGLK Ser tail
with his tag PGVDYTITVYAVTDWYNPDTHEYIYHTIPISINY RTEIDKPSQHHHHHH 156
E90 EGFR monomer core: EVVAATPTSLLISWYWEGLPYQYYRITYGETG E90 monomer
without N- GNSPVQEFTVPRDVNTATISGLKPGVDYTITVY terminal extension or
C- AVTDWYNPDTHEYIYHTIPISINYRT terminal tail 157 E90 EGFR monomer,
wherein X.sub.1EVVAATPTSLLISWYWEGLPYQYYRITYGET X.sub.1 is selected
from the group GGNSPVQEFTVPRDVNTATISGLKPGVDYTITV consisting of SEQ
ID NOs: 69- YAVTDWYNPDTHEYIYHTIPISINYRTX.sub.2 77 and X.sub.2 is
selected from the group consisting of SEQ ID NOs: 9, 10, or 78-81;
in exemplary emobidments, X.sub.1 is SEQ ID NO: 69 or 71 and
X.sub.2 is SEQ ID NO: 9 or 10; may optionally comprise a his tag
158 BC loop sequence from EGFR SWYWEGLPYQ binder E90 159 DE loop
sequence from EGFR PRDVNT binder E90 160 FG loop sequence from EGFR
TDWYNPDTHEYIYHTIP binder E90 161 BC loop sequence from EGFR
X.sub.gYWEGLPYQX.sub.h binder E90, wherein X is any amino acid and
g and h are independently selected from 0 to 5 amino acids 162 DE
loop sequence from EGFR X.sub.iRDVNX.sub.j binder E90, wherein X is
any amino acid and i and j are independently selected from 0 to 5
amino acids 163 FG loop sequence from EGFR
X.sub.kDWYNPDTHEYIYHTIX.sub.l binder E90, wherein X is any amino
acid and k and l are independently selected from 0 to 5 amino acids
164 E90-GS10-I1: E/I tandem MGVSDVPRDLEVVAATPTSLLISWYWEGLPYQ having
E90 (with N-terminal YYRITYGETGGNSPVQEFTVPRDVNTATISGLK extension (N
+ 10) and a short PGVDYTITVYAVTDWYNPDTHEYIYHTIPISINY tail) fused
via GS10 linker RTEIDKGSGSGSGSGSGSGSGSGSGSVSDVPR (GS10 is SEQ ID
NO: 11) to DLEVVAATPTSLLISWSARLKVARYYRITYGET I1 (with N-terminal
extension GGNSPVQEFTVPKNVYTATISGLKPGVDYTITV (N + 8) and Cys tail)
with a 6X YAVTRFRDYQPISINYRTEIDKPCQHHHHHH His tag (SEQ ID NO: 487)
165 E90-GS10-I1 core, wherein X.sub.1
X.sub.1EVVAATPTSLLISWYWEGLPYQYYRITYGET is optional and when present
is GGNSPVQEFTVPRDVNTATISGLKPGVDYTITV selected from the group
YAVTDWYNPDTHEYIYHTIPISINYRTEIDKGSG consisting of SEQ ID NOs: 69-
SGSGSGSGSGSGSGSGSVSDVPRDLEVVAATP 77, X.sub.2 is optional and when
TSLLISWSARLKVARYYRITYGETGGNSPVQEF
present is selected from the TVPKNVYTATISGLKPGVDYTITVYAVTRFRDY
group consisting of SEQ ID QPISINYRTX.sub.2 NOs: 9, 10, or 78-81,
and n = 3, 6 or 9; in exemplary embodiments, X.sub.1 is SEQ ID NO:
69 or 71 and X.sub.2 is SEQ ID NO: 9 or 10 166 E90-(PA).sub.n-I1
core, wherein X1 X.sub.1EVVAATPTSLLISWYWEGLPYQYYRITYGET is optional
and when present is GGNSPVQEFTVPRDVNTATISGLKPGVDYTITV selected from
the group YAVTDWYNPDTHEYIYHTIPISINYRTE(PA).sub.nVS consisting of
SEQ ID NOs: 69- DVPRDLEVVAATPTSLLISWSARLKVARYYRIT 77, X.sub.2 is
optional and when YGETGGNSPVQEFTVPKNVYTATISGLKPGVD present is
selected from the YTITVYAVTRFRDYQPISINYRTX.sub.2 group consisting
of SEQ ID NOs: 9, 10, or 78-81, and n = 3, 6 or 9; in exemplary
embodiments, X.sub.1 is SEQ ID NO: 69 or 71 and X.sub.2 is SEQ ID
NO: 9 or 10 167 I1-GS10-E90: I/E tandem
MGVSDVPRDLEVVAATPTSLLISWSARLKVAR having I1 (with N-terminal
YYRITYGETGGNSPVQEFTVPKNVYTATISGLK extension (N + 10) and a short
PGVDYTITVYAVTRFRDYQPISINYRTEIDKGSG tail) fused via GS10 linker
SGSGSGSGSGSGSGSGSVSDVPRDLEVVAATP (GS10 is SEQ ID NO: 11) to
TSLLISWYWEGLPYQYYRITYGETGGNSPVQEF E90 (with N-terminal
TVPRDVNTATISGLKPGVDYTITVYAVTDWYN extension (N + 8) and Cys tail)
PDTHEYIYHTIPISINYRTEIDKPCQHHHHHH with a 6X His tag (SEQ ID NO: 487)
168 I1-GS10-E90 core, wherein X.sub.1
X.sub.1EVVAATPTSLLISWSARLKVARYYRITYGET is optional and when present
is GGNSPVQEFTVPKNVYTATISGLKPGVDYTITV selected from the group
YAVTRFRDYQPISINYRTEIDKGSGSGSGSGSG consisting of SEQ ID NOs: 69-
SGSGSGSGSVSDVPRDLEVVAATPTSLLISWY 77, X.sub.2 is optional and when
WEGLPYQYYRITYGETGGNSPVQEFTVPRDVN present is selected from the
TATISGLKPGVDYTITVYAVTDWYNPDTHEYIY group consisting of SEQ ID
HTIPISINYRTX.sub.2 NOs: 9, 10, or 78-81, and n = 3, 6 or 9; in
exemplary embodiments, X.sub.1 is SEQ ID NO: 69 or 71 and X.sub.2
is SEQ ID NO: 9 or 10 169 I1-(PA).sub.n-E90 core, wherein X.sub.1
X.sub.1EVVAATPTSLLISWSARLKVARYYRITYGET is optional and when present
is GGNSPVQEFTVPKNVYTATISGLKPGVDYTITV selected from the group
YAVTRFRDYQPISINYRTE(PA).sub.nVSDVPRDLEV consisting of SEQ ID NOs:
69- VAATPTSLLISWYWEGLPYQYYRITYGETGGN 77, X.sub.2 is optional and
when SPVQEFTVPRDVNTATISGLKPGVDYTITVYAV present is selected from the
TDWYNPDTHEYIYHTIPISINYRTX.sub.2 group consisting of SEQ ID NOs: 9,
10, or 78-81, and n = 3, 6 or 9; in exemplary embodiments, X.sub.1
is SEQ ID NO: 69 or 71 and X.sub.2 is SEQ ID NO: 9 or 10 170 E96
EGFR monomer with N- MGVSDVPRDLEVVAATPTSLLISWASNRGTYQ terminal
extension (N + 10) and YYRITYGETGGNSPVQEFTVPGGVSTATISGLK Ser tail
with his tag PGVDYTITVYAVTDAFNPTTHEYNYFTTPISINY RTEIDKPSQHHHHHH 171
E96 EGFR monomer core: EVVAATPTSLLISWASNRGTYQYYRITYGETGG E96
monomer without N- NSPVQEFTVPGGVSTATISGLKPGVDYTITVYA terminal
extension or C- VTDAFNPTTHEYNYFTTPISINYRT terminal tail 172 E96
EGFR monomer, wherein X.sub.1EVVAATPTSLLISWASNRGTYQYYRITYGET
X.sub.1 is selected from the group
GGNSPVQEFTVPGGVSTATISGLKPGVDYTITV consisting of SEQ ID NOs: 69-
YAVTDAFNPTTHEYNYFTTPISINYRTX.sub.2 77 and X.sub.2 is selected from
the group consisting of SEQ ID NOs: 9, 10, or 78-81; in exemplary
emobidments, X.sub.1 is SEQ ID NO: 69 or 71 and X.sub.2 is SEQ ID
NO: 9 or 10; may optionally comprise a his tag 173 BC loop sequence
from EGFR SWASNRGTYQ binder E96 174 DE loop sequence from EGFR
PGGVST binder E96 175 FG loop sequence from EGFR TDAFNPTTHEYNYFTTP
binder E96 176 BC loop sequence from EGFR X.sub.gASNRGTYQX.sub.h
binder E96, wherein X is any amino acid and g and h are
independently selected from 0 to 5 amino acids 177 DE loop sequence
from EGFR X.sub.iGGVSX.sub.j binder E96, wherein X is any amino
acid and i and j are independently selected from 0 to 5 amino acids
178 FG loop sequence from EGFR X.sub.kDAFNPTTHEYNYFTTX.sub.l binder
E96, wherein X is any amino acid and k and l are independently
selected from 0 to 5 amino acids 179 E96-GS10-I1: E/I tandem
MGVSDVPRDLEVVAATPTSLLISWASNRGTYQ having E96 (with N-terminal
YYRITYGETGGNSPVQEFTVPGGVSTATISGLK extension (N + 10) and a short
PGVDYTITVYAVTDAFNPTTHEYNYFTTPISINY tail) fused via GS10 linker
RTEIDKGSGSGSGSGSGSGSGSGSGSVSDVPR (GS10 is SEQ ID NO: 11) to
DLEVVAATPTSLLISWSARLKVARYYRITYGET I1 (with N-terminal extension
GGNSPVQEFTVPKNVYTATISGLKPGVDYTITV (N + 8) and Cys tail) with a 6X
YAVTRFRDYQPISINYRTEIDKPCQHHHHHH His tag (SEQ ID NO: 487) 180
E96-GS10-I1 core, wherein X.sub.1
X.sub.1EVVAATPTSLLISWASNRGTYQYYRITYGET is optional and when present
is GGNSPVQEFTVPGGVSTATISGLKPGVDYTITV selected from the group
YAVTDAFNPTTHEYNYFTTPISINYRTEIDKGSG consisting of SEQ ID NOs: 69-
SGSGSGSGSGSGSGSGSVSDVPRDLEVVAATP 77, X.sub.2 is optional and when
TSLLISWSARLKVARYYRITYGETGGNSPVQEF present is selected from the
TVPKNVYTATISGLKPGVDYTITVYAVTRFRDY group consisting of SEQ ID
QPISINYRTX.sub.2 NOs: 9, 10, or 78-81, and n = 3, 6 or 9; in
exemplary embodiments, X.sub.1 is SEQ ID NO: 69 or 71 and X.sub.2
is SEQ ID NO: 9 or 10 181 E96-(PA).sub.n-I1 core, wherein X1
X.sub.1EVVAATPTSLLISWASNRGTYQYYRITYGET is optional and when present
is GGNSPVQEFTVPGGVSTATISGLKPGVDYTITV selected from the group
YAVTDAFNPTTHEYNYFTTPISINYRTE(PA).sub.nVS consisting of SEQ ID NOs:
69- DVPRDLEVVAATPTSLLISWSARLKVARYYRIT 77, X.sub.2 is optional and
when YGETGGNSPVQEFTVPKNVYTATISGLKPGVD present is selected from the
YTITVYAVTRFRDYQPISINYRTX.sub.2 group consisting of SEQ ID NOs: 9,
10, or 78-81, and n = 3, 6 or 9; in exemplary embodiments, X.sub.1
is SEQ ID NO: 69 or 71 and X.sub.2 is SEQ ID NO: 9 or 10 182
I1-GS10-E96: I/E tandem MGVSDVPRDLEVVAATPTSLLISWSARLKVAR having I1
(with N-terminal YYRITYGETGGNSPVQEFTVPKNVYTATISGLK extension (N +
10) and a short PGVDYTITVYAVTRFRDYQPISINYRTEIDKGSG tail) fused via
GS10 linker SGSGSGSGSGSGSGSGSVSDVPRDLEVVAATP (GS10 is SEQ ID NO:
11) to TSLLISWASNRGTYQYYRITYGETGGNSPVQEF E96 (with N-terminal
TVPGGVSTATISGLKPGVDYTITVYAVTDAFNP extension (N + 8) and Cys tail)
TTHEYNYFTTPISINYRTEIDKPCQHHHHHH with a 6X His tag (SEQ ID NO: 487)
183 I1-GS10-E96 core, wherein X.sub.1
X.sub.1EVVAATPTSLLISWSARLKVARYYRITYGET is optional and when present
is GGNSPVQEFTVPKNVYTATISGLKPGVDYTITV selected from the group
YAVTRFRDYQPISINYRTEIDKGSGSGSGSGSG consisting of SEQ ID NOs: 69-
SGSGSGSGSVSDVPRDLEVVAATPTSLLISWAS 77, X.sub.2 is optional and when
NRGTYQYYRITYGETGGNSPVQEFTVPGGVST present is selected from the
ATISGLKPGVDYTITVYAVTDAFNPTTHEYNYF group consisting of SEQ ID
TTPISINYRTX.sub.2 NOs: 9, 10, or 78-81, and n = 3, 6 or 9; in
exemplary embodiments, X.sub.1 is SEQ ID NO: 69 or 71 and X.sub.2
is SEQ ID NO: 9 or 10 184 I1-(PA).sub.n-E96 core, wherein X.sub.1
X.sub.1EVVAATPTSLLISWSARLKVARYYRITYGET is optional and when present
is GGNSPVQEFTVPKNVYTATISGLKPGVDYTITV selected from the group
YAVTRFRDYQPISINYRTE(PA).sub.nVSDVPRDLEV consisting of SEQ ID NOs:
69- VAATPTSLLISWASNRGTYQYYRITYGETGGNS 77, X.sub.2 is optional and
when PVQEFTVPGGVSTATISGLKPGVDYTITVYAVT present is selected from the
DAFNPTTHEYNYFTTPISINYRTX.sub.2 group consisting of SEQ ID NOs: 9,
10, or 78-81, and n = 3, 6 or 9; in exemplary embodiments, X.sub.1
is SEQ ID NO: 69 or 71 and X2 is SEQ ID NO: 9 or 10 185 E105 EGFR
monomer with N- MGVSDVPRDLEVVAATPTSLLISWDAPTSRYQ terminal extension
(N + 10) and YYRITYGETGGNSPVQEFTVPGGLSTATISGLKP Ser tail with his
tag GVDYTITVYAVTDYKPHADGPHTYHESPISINY RTEIDKPSQHHHHHH 186 E105 EGFR
monomer core: EVVAATPTSLLISWDAPTSRYQYYRITYGETGG E105 monomer
without N- NSPVQEFTVPGGLSTATISGLKPGVDYTITVYA terminal extension or
C- VTDYKPHADGPHTYHESPISINYRT terminal tail 187 E105 EGFR monomer,
X.sub.1EVVAATPTSLLISWDAPTSRYQYYRITYGET wherein X.sub.1 is selected
from the GGNSPVQEFTVPGGLSTATISGLKPGVDYTITV group consisting of SEQ
ID YAVTDYKPHADGPHTYHESPISINYRTX.sub.2 NOs: 69-77 and X.sub.2 is
selected from the group consisting of SEQ ID NOs: 9, 10, or 78-81;
in exemplary emobidments, X.sub.1 is SEQ ID NO: 69 or 71 and
X.sub.2 is SEQ ID NO: 9 or 10; may optionally comprise a his tag
188 BC loop sequence from EGFR SWDAPTSRYQ binder E105 189 DE loop
sequence from EGFR PGGLST binder E105 117 FG loop sequence from
EGFR TDYKPHADGPHTYHESP binder E105 190 BC loop sequence from EGFR
X.sub.gDAPTSRYQX.sub.h binder E105, wherein X is any amino acid and
g and h are independently selected from 0 to 5 amino acids 191 DE
loop sequence from EGFR X.sub.iGGLSX.sub.j binder E105, wherein X
is any amino acid and i and j are independently selected from 0 to
5 amino acids 139 FG loop sequence from EGFR
X.sub.kDYKPHADGPHTYHESX.sub.l binder E105, wherein X is any amino
acid and k and l are independently selected from 0 to 5 amino acids
192 E105-GS10-I1: E/I tandem MGVSDVPRDLEVVAATPTSLLISWDAPTSRYQ
having E105 (with N-terminal YYRITYGETGGNSPVQEFTVPGGLSTATISGLKP
extension (N + 10) and a short GVDYTITVYAVTDYKPHADGPHTYHESPISINY
tail) fused via GS10 linker RTEIDKGSGSGSGSGSGSGSGSGSGSVSDVPR (GS10
is SEQ ID NO: 11) to DLEVVAATPTSLLISWSARLKVARYYRITYGET I1 (with
N-terminal extension GGNSPVQEFTVPKNVYTATISGLKPGVDYTITV (N + 8) and
Cys tail) with a 6X YAVTRFRDYQPISINYRTEIDKPCQHHHHHH His tag (SEQ ID
NO: 487) 193 E105-GS10-I1 core, wherein
X.sub.1EVVAATPTSLLISWDAPTSRYQYYRITYGET X.sub.1 is optional and when
GGNSPVQEFTVPGGLSTATISGLKPGVDYTITV present is selected from the
YAVTDYKPHADGPHTYHESPISINYRTEIDKGS group consisting of SEQ ID
GSGSGSGSGSGSGSGSGSVSDVPRDLEVVAAT NOs: 69-77, X.sub.2 is optional
and PTSLLISWSARLKVARYYRITYGETGGNSPVQE when present is selected from
FTVPKNVYTATISGLKPGVDYTITVYAVTRFRD the group consisting of SEQ
YQPISINYRTX.sub.2 ID NOs: 9, 10, or 78-81, and n = 3, 6 or 9; in
exemplary embodiments, X.sub.1 is SEQ ID
NO: 69 or 71 and X.sub.2 is SEQ ID NO: 9 or 10 194
E105-(PA).sub.n-I1 core, wherein
X.sub.1EVVAATPTSLLISWDAPTSRYQYYRITYGET X1 is optional and when
GGNSPVQEFTVPGGLSTATISGLKPGVDYTITV present is selected from the
YAVTDYKPHADGPHTYHESPISINYRTE(PA).sub.nV group consisting of SEQ ID
SDVPRDLEVVAATPTSLLISWSARLKVARYYRI NOs: 69-77, X.sub.2 is optional
and TYGETGGNSPVQEFTVPKNVYTATISGLKPGVD when present is selected from
YTITVYAVTRFRDYQPISINYRTX.sub.2 the group consisting of SEQ ID NOs:
9, 10, or 78-81, and n = 3, 6 or 9; in exemplary embodiments,
X.sub.1 is SEQ ID NO: 69 or 71 and X.sub.2 is SEQ ID NO: 9 or 10
195 I1-GS10-E105: I/E tandem MGVSDVPRDLEVVAATPTSLLISWSARLKVAR
having I1 (with N-terminal YYRITYGETGGNSPVQEFTVPKNVYTATISGLK
extension (N + 10) and a short PGVDYTITVYAVTRFRDYQPISINYRTEIDKGSG
tail) fused via GS10 linker SGSGSGSGSGSGSGSGSVSDVPRDLEVVAATP (GS10
is SEQ ID NO: 11) to TSLLISWDAPTSRYQYYRITYGETGGNSPVQEF E105 (with
N-terminal TVPGGLSTATISGLKPGVDYTITVYAVTDYKPH extension (N + 8) and
Cys tail) ADGPHTYHESPISINYRTEIDKPCQHHHHHH with a 6X His tag (SEQ ID
NO: 487) 196 I1-GS10-E105 core, wherein
X.sub.1EVVAATPTSLLISWSARLKVARYYRITYGET X.sub.1 is optional and when
GGNSPVQEFTVPKNVYTATISGLKPGVDYTITV present is selected from the
YAVTRFRDYQPISINYRTEIDKGSGSGSGSGSG group consisting of SEQ ID
SGSGSGSGSVSDVPRDLEVVAATPTSLLISWDA NOs: 69-77, X.sub.2 is optional
and PTSRYQYYRITYGETGGNSPVQEFTVPGGLSTA when present is selected from
TISGLKPGVDYTITVYAVTDYKPHADGPHTYH the group consisting of SEQ
ESPISINYRTX.sub.2 ID NOs: 9, 10, or 78-81, and n = 3, 6 or 9; in
exemplary embodiments, X.sub.1 is SEQ ID NO: 69 or 71 and X.sub.2
is SEQ ID NO: 9 or 10 197 I1-(PA).sub.n-E105 core, wherein
X.sub.1EVVAATPTSLLISWSARLKVARYYRITYGET X.sub.1 is optional and when
GGNSPVQEFTVPKNVYTATISGLKPGVDYTITV present is selected from the
YAVTRFRDYQPISINYRTE(PA).sub.nVSDVPRDLEV group consisting of SEQ ID
VAATPTSLLISWDAPTSRYQYYRITYGETGGNS NOs: 69-77, X.sub.2 is optional
and PVQEFTVPGGLSTATISGLKPGVDYTITVYAVT when present is selected from
DYKPHADGPHTYHESPISINYRTX.sub.2 the group consisting of SEQ ID NOs:
9, 10, or 78-81, and n = 3, 6 or 9; in exemplary embodiments,
X.sub.1 is SEQ ID NO: 69 or 71 and X.sub.2 is SEQ ID NO: 9 or 10
198 E112 EGFR monomer with N- MGVSDVPRDLEVVAATPTSLLISWDAGAVTYQ
terminal extension (N + 10) and YYRITYGETGGNSPVQEFTVPGGVRTATISGLK
Ser tail with his tag PGVDYTITVYAVTDYKPHADGPHTYHEYPISIN
YRTEIDKPSQHHHHHH 199 E112 EGFR monomer core:
EVVAATPTSLLISWDAGAVTYQYYRITYGETG E112 monomer without N-
GNSPVQEFTVPGGVRTATISGLKPGVDYTITVY terminal extension or C-
AVTDYKPHADGPHTYHEYPISINYRT terminal tail 200 E112 EGFR monomer,
X.sub.1EVVAATPTSLLISWDAGAVTYQYYRITYGET wherein X.sub.1 is selected
from the GGNSPVQEFTVPGGVRTATISGLKPGVDYTITV group consisting of SEQ
ID YAVTDYKPHADGPHTYHEYPISINYRTX.sub.2 NOs: 69-77 and X.sub.2 is
selected from the group consisting of SEQ ID NOs: 9, 10, or 78-81;
in exemplary emobidments, X.sub.1 is SEQ ID NO: 69 or 71 and
X.sub.2 is SEQ ID NO: 9 or 10; may optionally comprise a his tag
201 BC loop sequence from EGFR SWDAGAVTYQ binder E112 110 DE loop
sequence from EGFR PGGVRT binder E112 202 FG loop sequence from
EGFR TDYKPHADGPHTYHEYP binder E112 203 BC loop sequence from EGFR
X.sub.gDAGAVTYQX.sub.h binder E112, wherein X is any amino acid and
g and h are independently selected from 0 to 5 amino acids 135 DE
loop sequence from EGFR X.sub.iGGVRX.sub.j binder E112, wherein X
is any amino acid and i and j are independently selected from 0 to
5 amino acids 204 FG loop sequence from EGFR
X.sub.kDYKPHADGPHTYHEYX.sub.l binder E112, wherein X is any amino
acid and k and l are independently selected from 0 to 5 amino acids
205 E112-GS10-I1: E/I tandem MGVSDVPRDLEVVAATPTSLLISWDAGAVTYQ
having E112 (with N-terminal YYRITYGETGGNSPVQEFTVPGGVRTATISGLK
extension (N + 10) and a short PGVDYTITVYAVTDYKPHADGPHTYHEYPISIN
tail) fused via GS10 linker YRTEIDKGSGSGSGSGSGSGSGSGSGSVSDVP (GS10
is SEQ ID NO: 11) to RDLEVVAATPTSLLISWSARLKVARYYRITYGE I1 (with
N-terminal extension TGGNSPVQEFTVPKNVYTATISGLKPGVDYTIT (N + 8) and
Cys tail) with a 6X VYAVTRFRDYQPISINYRTEIDKPCQHHHHHH His tag (SEQ
ID NO: 487) 206 E112-GS10-I1 core, wherein
X.sub.1EVVAATPTSLLISWDAGAVTYQYYRITYGET X.sub.1 is optional and when
GGNSPVQEFTVPGGVRTATISGLKPGVDYTITV present is selected from the
YAVTDYKPHADGPHTYHEYPISINYRTEIDKGS group consisting of SEQ ID
GSGSGSGSGSGSGSGSGSVSDVPRDLEVVAAT NOs: 69-77, X.sub.2 is optional
and PTSLLISWSARLKVARYYRITYGETGGNSPVQE when present is selected from
FTVPKNVYTATISGLKPGVDYTITVYAVTRFRD the group consisting of SEQ
YQPISINYRTX.sub.2 ID NOs: 9, 10, or 78-81, and n = 3, 6 or 9; in
exemplary embodiments, X.sub.1 is SEQ ID NO: 69 or 71 and X.sub.2
is SEQ ID NO: 9 or 10 207 E112-(PA).sub.n-I1 core, wherein
X.sub.1EVVAATPTSLLISWDAGAVTYQYYRITYGET X1 is optional and when
GGNSPVQEFTVPGGVRTATISGLKPGVDYTITV present is selected from the
YAVTDYKPHADGPHTYHEYPISINYRTE(PA).sub.nV group consisting of SEQ ID
SDVPRDLEVVAATPTSLLISWSARLKVARYYRI NOs: 69-77, X.sub.2 is optional
and TYGETGGNSPVQEFTVPKNVYTATISGLKPGVD when present is selected from
YTITVYAVTRFRDYQPISINYRTX.sub.2 the group consisting of SEQ ID NOs:
9, 10, or 78-81, and n = 3, 6 or 9; in exemplary embodiments,
X.sub.1 is SEQ ID NO: 69 or 71 and X.sub.2 is SEQ ID NO: 9 or 10
208 I1-GS10-E112: I/E tandem MGVSDVPRDLEVVAATPTSLLISWSARLKVAR
having I1 (with N-terminal YYRITYGETGGNSPVQEFTVPKNVYTATISGLK
extension (N + 10) and a short PGVDYTITVYAVTRFRDYQPISINYRTEIDKGSG
tail) fused via GS10 linker SGSGSGSGSGSGSGSGSVSDVPRDLEVVAATP (GS10
is SEQ ID NO: 11) to TSLLISWDAGAVTYQYYRITYGETGGNSPVQEF E112 (with
N-terminal TVPGGVRTATISGLKPGVDYTITVYAVTDYKPH extension (N + 8) and
Cys tail) ADGPHTYHEYPISINYRTEIDKPCQHHHHHH with a 6X His tag (SEQ ID
NO: 487) 209 I1-GS10-E112 core, wherein
X.sub.1EVVAATPTSLLISWSARLKVARYYRITYGET X.sub.1 is optional and when
GGNSPVQEFTVPKNVYTATISGLKPGVDYTITV present is selected from the
YAVTRFRDYQPISINYRTEIDKGSGSGSGSGSG group consisting of SEQ ID
SGSGSGSGSVSDVPRDLEVVAATPTSLLISWDA NOs: 69-77, X.sub.2 is optional
and GAVTYQYYRITYGETGGNSPVQEFTVPGGVRT when present is selected from
ATISGLKPGVDYTITVYAVTDYKPHADGPHTY the group consisting of SEQ
HEYPISINYRTX.sub.2 ID NOs: 9, 10, or 78-81, and n = 3, 6 or 9; in
exemplary embodiments, X.sub.1 is SEQ ID NO: 69 or 71 and X.sub.2
is SEQ ID NO: 9 or 10 210 I1-(PA).sub.n-E112 core, wherein
X.sub.1EVVAATPTSLLISWSARLKVARYYRITYGET X.sub.1 is optional and when
GGNSPVQEFTVPKNVYTATISGLKPGVDYTITV present is selected from the
YAVTRFRDYQPISINYRTE(PA).sub.nVSDVPRDLEV group consisting of SEQ ID
VAATPTSLLISWDAGAVTYQYYRITYGETGGN NOs: 69-77, X.sub.2 is optional
and SPVQEFTVPGGVRTATISGLKPGVDYTITVYAV when present is selected from
TDYKPHADGPHTYHEYPISINYRTX.sub.2 the group consisting of SEQ ID NOs:
9, 10, or 78-81, and n = 3, 6 or 9; in exemplary embodiments,
X.sub.1 is SEQ ID NO: 69 or 71 and X.sub.2 is SEQ ID NO: 9 or 10
211 I1-GSGCGS8-E5: I/E tandem MGVSDVPRDLEVVAATPTSLLISWSARLKVAR
having I1 (with N-terminal YYRITYGETGGNSPVQEFTVPKNVYTATISGLK
extension (N + 10) and a PGVDYTITVYAVTRFRDYQPISINYRTEIEKGSG
modified short tail) fused via CGSGSGSGSGSGSGSGSVSDVPRDLEVVAATP
GSGCGS8 linker (GSGCGS8 TSLLISWWAPVDRYQYYRITYGETGGNSPVQE is SEQ ID
NO: 218) to E5 FTVPRDVYTATISGLKPGVDYTITVYAVTDYKP (with N-terminal
extension HADGPHTYHESPISINYRTEHHHHHH (N + 8) and an E tail) with an
optional 6X His tag (SEQ ID NO: 487) 212 I1-GS10-E5-GSGC: I/E
MGVSDVPRDLEVVAATPTSLLISWSARLKVAR tandem having I1 (with N-
YYRITYGETGGNSPVQEFTVPKNVYTATISGLK terminal extension (N + 10) and
PGVDYTITVYAVTRFRDYQPISINYRTEIEKGSG a modified short tail) fused via
SGSGSGSGSGSGSGSGSVSDVPRDLEVVAATP GS10 linker (GS10 is SEQ ID
TSLLISWWAPVDRYQYYRITYGETGGNSPVQE NO: 11) to E5 (with N-
FTVPRDVYTATISGLKPGVDYTITVYAVTDYKP terminal extension (N + 8) and a
HADGPHTYHESPISINYRTEGSGCHHHHHH modified Cys tail) with an optional
6X His tag (SEQ ID NO: 487) 213 I1 (S62C)-GS10-E5: I/E tandem
having I1 (with N- terminal extension (N + 10), an S62C
substitution (boxed), and a modified short tail) fused via GS10
linker (GS10 is SEQ ID NO: 11) to E5 (with N- terminal extension (N
+ 8) and an E tail) with an optional 6X His tag (SEQ ID NO: 487);
position 62 refers to the amino acid corresponding to position 62
of SEQ ID NO: 1 ##STR00001## 214 I1-GS10-E5(S62C): I/E tandem
having I1 (with N- terminal extension (N + 10) and a modified short
tail) fused via GS10 linker (GS10 is SEQ ID NO: 11) to E5 (with N-
terminal extension (N + 8), an S62C substitution (boxed), and an E
tail) with an optional 6X His tag (SEQ ID NO: 487); position 62
refers to the amino acid corresponding to position 62 of SEQ ID NO:
1 ##STR00002## 215 I1(S91C)-GS10-E5: I/E tandem having I1 (with N-
terminal extension (N + 10), an S91C substitution (boxed), and a
modified short tail) fused via GS10 linker (GS10 is SEQ ID NO: 11)
to E5 (with N- terminal extension (N + 8) and an E tail) with an
optional 6X His tag (SEQ ID NO: 487); position 91 refers to the
amino acid corresponding to position 91 of SEQ ID NO: 1
##STR00003## 216 I1-GS10-E5(S91C): I/E tandem having I1 (with N-
terminal extension (N + 10) and a modified short tail) fused via
GS10 linker (GS10 is SEQ ID NO: 11) to E5 (with N- terminal
extension (N + 8), an S91C substitution (boxed), and an E tail)
with an optional 6X His tag (SEQ ID NO: 487); position 91 refers to
the amino acid corresponding to position 91 of SEQ ID NO: 1
##STR00004## 217 Modified short tail EIEK 218 GSGCGS8 Linker
GSGCGSGSGSGSGSGSGSGS
Example 1
In Cell Western Assay to Screen for EGFR Activity
In Cell Western assays were developed to screen various single
.sup.10Fn3 clones for the ability to inhibit EGFR activity in order
to identify those that could be linked with IGF1R .sup.10Fn3
binders to construct E/I binders. In Cell Western assays were also
used to screen and determine relative potency of specific E/I
.sup.10Fn3 binders. Two In Cell Western assays were developed to
measure 1) inhibition of EGF-stimulated EGFR phosphorylation or 2)
inhibition of EGF-stimulated ERK phosphorylation. Cells were seeded
into poly-D-lysine coated 96-well microtiter plates (Becton
Dickinson, Franklin Lakes, N.J.) at 24,000 cells/well for A431
epidermoid carcinoma or FaDu head & neck carcinoma cells and
allowed to adhere overnight. Cells were washed once and then
incubated for 24 hours in serum free media. Serial dilutions of the
.sup.10Fn3-based binders were next applied to the cells and
incubated for 2-3 hours prior to stimulation with 100 ng/ml EGF for
10 minutes. Following stimulation, cells were fixed for 20 minutes
in PBS containing 3.7% formaldehyde and then permeabilized in PBS
containing 0.1% triton-X-100 for 15 minutes. Cells were blocked for
one hour in Odyssey blocker (Li-Cor Biosciences, Lincoln, Nebr.)
and incubated with antibodies to detect either EGFR phosphorylated
on tyrosine 1068 (Cell Signaling, Beverly, Mass.) and .beta.-actin
(Sigma, St. Louis, Mo.) or pERK (MAP kinase phosphorylated on
tyrosine 202/threonine 204) and total ERK (Santa Cruz
Biotechnology, Santa Cruz, Calif.). After washing three times in
PBS containing 0.1% tween-20, secondary antibodies were added
(Invitrogen, Carlsbad, Calif. or Rockland, Gilbertsville, Pa.).
Cells were washed three times in PBS containing 0.1% tween-20 and
imaged on a Li-Cor Odyssey Infrared Imaging System (Li-Cor
Biosciences, Lincoln, Nebr.). Each clone was assayed in duplicate
or triplicate and values were normalized to .beta.-actin for the
pEGFR assay and total ERK for the pERK assay. IC50 values were
calculated from linear regression analysis of percent inhibition of
maximum signal minus background.
Results yielded various .sup.10Fn3 clones that had ability to
inhibit activity of EGFR, and showed that certain specific E/I
.sup.10Fn3 binders possessed similar activity to the example shown
in FIG. 9.
Example 2
Expression of .sup.10Fn3-Based Binders
E/I binders were produced by covalently linking an EGFR-binding
.sup.10Fn3 to an IGFIR-binding .sup.10Fn3 using a glycine-serine
linker, thereby generating .sup.10Fn3 dimers, wherein each
.sup.10Fn3 domain binds to a different target. The IGFIR-binding
.sup.10Fn3 (I1) was previously described as SEQ ID NO: 226 in PCT
Publication No. WO 2008/066752. Two novel EGFR-binding .sup.10Fn3
(E2 and E1) were identified by screening an RNA-protein fusion
library, as described in PCT Publication No. WO 2008/066752, for
binders to EGFR-Fc (R&D Systems, Minneapolis, Minn.). The
following examples describe results using a variety of His-tagged
E/I .sup.10Fn3-based binders (non-pegylated): E2-GS10-I1 (SEQ ID
NO: 25), E1-GS10-I1 (SEQ ID NO: 31), I1-GS10-E1 (SEQ ID NO: 28),
and I1-GS10-E2 (SEQ ID NO: 22).
The following examples also describe results with the following
pegylated, His-tagged E/I .sup.10Fn3-based binders: E1-GS10-I1 (SEQ
ID NO: 55), E2-GS10-I1 (SEQ ID NO: 56), E3-GS10-I1 (SEQ ID NO: 53),
I1-GS10-E1 (SEQ ID NO: 57), I1-GS10-E2 (SEQ ID NO: 58), I1-GS10-E3
(SEQ ID NO: 54), E4-GS10-I1 (SEQ ID NO: 120), I1-GS10-E4 (SEQ ID
NO: 124), E5-GS10-I1 (SEQ ID NO: 128), I1-GS10-E5 (SEQ ID NO: 132),
E85-GS10-I1 (SEQ ID NO: 149), I1-GS10-E85 (SEQ ID NO: 152),
E90-GS10-I1 (SEQ ID NO: 164), E96-GS10-I1 (SEQ ID NO: 179),
E105-GS10-I1 (SEQ ID NO: 192), I1-GS10-E105 (SEQ ID NO: 195),
E112-GS10-I1 (SEQ ID NO: 205), I1-GS10-E112 (SEQ ID NO: 208),
I1-GSGCGS8-E5 (SEQ ID NO: 211), I1-GS10-E5-GSGC (SEQ ID NO: 212),
I1(S62C)-GS10-E5 (SEQ ID NO: 213), I1-GS10-E5(S62C) (SEQ ID NO:
214), I1(S91C)-GS10-E5 (SEQ ID NO: 215), and I1-GS10-E5(S91C) (SEQ
ID NO: 216).
The examples also describe results using a His-tagged IGFRIR
.sup.10Fn3-based binder, I1 (SEQ ID NO: 4), and ten His-tagged EGFR
.sup.10Fn3-based binders, E2 (SEQ ID NO: 6), E1 (SEQ ID NO: 8), E3
(SEQ ID NO: 52), E4 (SEQ ID NO: 107), E5 (SEQ ID NO: 113, wherein
X=Ser and with a His tag at the C-terminus), E5 pegylated (SEQ ID
NO: 113, wherein X=Cys and with a His tag at the C-terminus), E85
(SEQ ID NO: 140), E90 (SEQ ID NO: 155), E96 (SEQ ID NO: 170), E105
(SEQ ID NO: 185), and E112 (SEQ ID NO: 198). Examples 32 also
describes a variety of E monomers having the sequences set forth in
FIG. 45 and including a His tag at the C-terminus.
The various .sup.10Fn3-based binders were purified using a high
throughput protein production process (HTPP). Selected binders were
cloned into the pET9d vector in order to generate His.sub.6 tag
(SEQ ID NO: 487) fusions. DNA was transformed into E. coli
HMS174(DE3), and cells were inoculated in 5 ml LB medium containing
50 .mu.g/mL kanamycin in a 24-well format and grown at 37.degree.
C. overnight. Fresh 5 ml LB medium (50 .mu.g/mL kanamycin) cultures
were prepared for inducible expression by aspirating 200 .mu.l from
the overnight culture and dispensing it into the appropriate well.
The cultures were grown at 37.degree. C. until A.sub.600 0.6-0.9.
After induction with 1 mM isopropyl-.beta.-thiogalactoside (IPTG),
the culture was grown for another 6 hours at 30.degree. C. and
harvested by centrifugation for 10 minutes at 3220.times.g at
4.degree. C. Cell pellets were frozen at 80.degree. C.
Cell pellets (in 24-well format) were lysed by resuspension in 450
.mu.l of Lysis buffer (50 mM NaH.sub.2PO.sub.4, 0.5 M NaCl,
1.times. Complete.TM. Protease Inhibitor Cocktail-EDTA free
(Roche), 1 mM PMSF, 10 mM CHAPS, 40 mM imidazole, 1 mg/ml lysozyme,
30 ug/ml DNAse, 2 .mu.g/ml aprotonin, pH 8.0) and shaken at room
temperature for 1 hour. Lysates were clarified and re-racked into a
96-well format by transfer into a 96-well Whatman GF/D Unifilter
fitted with a 96-well, 650 .mu.l catch plate and centrifuged for 5
minutes at 200.times.g. The clarified lysates were transferred to a
96-well Ni-Chelating Plate that had been equilibrated with
equilibration buffer (50 mM NaH.sub.2PO.sub.4, 0.5 M NaCl, 10 mM
CHAPS, 40 mM imidazole, pH 8.0) and incubated for 5 minutes.
Unbound material was removed by vacuum. The resin was washed
2.times.0.3 ml/well with Wash buffer #1 (50 mM NaH.sub.2PO.sub.4,
0.5 M NaCl, 5 mM CHAPS, 40 mM imidazole, pH 8.0) with each wash
removed by vacuum. Next, the resin was washed with 3.times.0.3
ml/well with PBS with each wash step removed by vacuum. Prior to
elution, each well was washed with 50 .mu.l Elution buffer (PBS+20
mM EDTA), incubated for 5 minutes, and the wash discarded by
vacuum. Protein was eluted by applying an additional 100 .mu.l of
Elution buffer to each well. After a 30 minute incubation at room
temperature, the plate(s) were centrifuged for 5 minutes at
200.times.g and eluted protein collected in 96-well catch plates
containing 5 .mu.l of 0.5 M MgCl.sub.2 affixed to the bottom of the
Ni-plates. Eluted protein was quantified using a BCA Protein assay
with SEQ ID NO: 2 as the protein standard.
HTPP yielded active .sup.10Fn3-based binders that were expressed in
a soluble form and purified from the soluble fraction of the
bacterial cytosol. FIG. 1 depicts an exemplary SDS-PAGE analysis
from one of the E/I .sup.10Fn3-based binders. SEC analysis on a
Superdex 200 5/150 GL in a mobile phase of 100 mM NaPO4, 100 mM
NaSO4, 150 mM NaCl, pH 6.8 (GE Healthcare) demonstrated
predominantly monomeric proteins (see Example 4).
In addition, midscale expression and purification of select
.sup.10Fn3-based binders was performed. The selected binders, fused
to a His.sub.6 tag (SEQ ID NO: 487), were cloned into a pET9d or
pET29 vector and expressed in E. coli HMS174(DE3) or BL212(DE3)
(EMD Biosciences, San Diego, Calif.) cells. 20 ml of an inoculum
culture (generated from a single plated colony) was used to
inoculate 1 liter of LB medium containing 50 .mu.g/mL kanamycin.
The culture was grown at 37.degree. C. until A.sub.600 0.6-1.0.
After induction with 1 mM isopropyl-.beta.-thiogalactoside (IPTG),
the culture was grown for another 6 hours at 30.degree. C.
Alternatively, expression was carried out at 18.degree. C. after
initial growth at 37.degree. C. using autoinduction media ("ONE"
medium, EMD Biosciences, San Diego, Calif.). Cell pellets were
harvested by centrifugation for 30 minutes at
.gtoreq.10,000.times.g at 4.degree. C. and frozen at 80.degree. C.
The cell pellet was resuspended in 25 mL of lysis buffer (20 mM
NaH.sub.2PO.sub.4, 0.5 M NaCl, 1.times. Complete.TM. Protease
Inhibitor Cocktail-EDTA free (Roche), pH 7.4) using an Ultra-turrax
homgenizer on ice. Cell lysis was achieved by high pressure
homogenization (.gtoreq.18,000 psi) using a Model M-110S
Microfluidizer (Microfluidics). The soluble fraction was separated
by centrifugation for 30 minutes at 23,300.times.g at 4.degree. C.
The supernatant was clarified via 0.45 .mu.m filter. The clarified
lysate was loaded onto a HisTrap column (GE) pre-equilibrated with
20 mM NaH.sub.2PO.sub.4, 0.5 M NaCl, pH 7.4. The column was then
washed with 25 column volumes of 20 mM NaH.sub.2PO.sub.4, 0.5 M
NaCl, pH 7.4, followed by 20 column volumes of 20 mM
NaH.sub.2PO.sub.4, 0.5 M NaCl, 25 mM imidazole, pH 7.4, and then 35
column volumes of 20 mM NaH.sub.2PO.sub.4, 0.5 M NaCl, 40 mM
imidazole, pH 7.4. Protein was eluted with 15 column volumes of 20
mM NaH.sub.2PO.sub.4, 0.5 M NaCl, 500 mM imidazole, pH 7.4,
fractions pooled based on absorbance at A.sub.280 and dialyzed
against 1.times.PBS, 50 mM Tris, 150 mM NaCl, pH 8.5 or 50 mM
NaOAc, 150 mM NaCl, pH4.5. Any precipitate was removed by filtering
at 0.22 .mu.m.
Midscale expression and purification yielded highly pure and active
proteins that were expressed in a soluble form and purified from
the soluble fraction of the bacterial cytosol. SEC analysis on a
Superdex 200 10/30GL in a mobile phase of 100 mM NaPO.sub.4, 100 mM
NaSO.sub.4, 150 mM NaCl, pH 6.8 (GE Healthcare) demonstrated
predominantly monomeric proteins (see Example 4).
Example 3
Pegylation of E/I .sup.10Fn3-Based Binders
Multi-valent fibronectin based scaffold proteins, such as E/I
.sup.10Fn3-based binders, can be pegylated with various sizes and
types of PEG. To allow for pegylation, the protein is typically
modified near the C-terminus by a single point mutation of an amino
acid, typically a serine, to a cysteine. PEGylation of the protein
at the single cysteine residue is accomplished through conjugation
with various maleimide-derivatized PEG forms by combining the
derivatized-PEG reagent with the protein solution and incubating.
Progress and confirmation of the PEGylation conjugation reaction
can be confirmed by SDS-PAGE and/or SE-HPLC methods that separate
the non-PEGylated protein from the PEGylated protein.
For example, the construct E2-GS10-I1 (SEQ ID NO: 25) was pegylated
by replacing a serine that was at position 221 with a cysteine. The
resulting construct, SEQ ID NO: 56, was then conjugated with a
maleimide-derivatized 40 kDa branched PEG (NOF America Corporation,
White Plains, N.Y.). The derivatized PEG reagent was mixed with the
protein construct in solution and incubated at pH 7.40 at Room
temperature until the reaction was complete, typically 30 minutes
or overnight at 4.degree. C. The pH was lowered to pH 4.5 or pH 5.0
by dialysis or rapid desalting using size exclusion column
chromotography into in 50 NaOAc, 150 mM NaCl buffer. The mixture of
products and excess reactants from the PEGylation reaction were
then loaded onto a cation exchange chromotography column at the
lowered pH and eluted with a 150 mM to 1 M NaCl gradient. Studies
to confirm the pegylation were also conducted as described in the
paragraph above. The conjugations can be performed with the His
tagged or the His-Tag free versions of the protein.
On occasions in which E coli endotoxin contamination needed to be
depleted in the sample, two methods used either separately or in
conjunction with one another were employed. The first was to wash
the cation exchange column with typically 5 column volumes NaOAc
buffer supplemented with 0.5% Triton X-100, followed by 20 column
volumes (or more) of the same buffer without Triton X-100.
Additionally or in place of this procedure, the protein was passed
very slowly through a Sartorius Sartobind.RTM. Q filter (Sartorius
Stedim Biotech Bohemia, New York).
Two of the E/I .sup.10Fn3-based binders, E2-GS10-I1-cys (with his)
(SEQ ID NO: 56) and E3-GS10-I1-Cys (with his) (SEQ ID NO: 53), were
pegylated using an alternative procedure. Five ml of an inoculum
culture of BL21(DE3) E. coli cells containing a T7 polymerase
driven pET29 plasmid encoding either E2-GS10-I1-cys (with his) or
E3-GS10-I1-Cys (with his), were generated from a single plated
colony and used to inoculate 1 liter of auto-induction media ("ONE"
medium, EMD Biosciences, San Diego, Calif.) containing 50 .mu.g/mL
kanamycin. Expression was carried out at 18.degree. C. after
initial growth at 37.degree. C. and harvested by centrifugation for
10 minutes at .about.10,000.times.g at 4.degree. C. Cell pellets
were frozen at 80.degree. C. The cell pellet was resuspended in 10
mL of lysis buffer (20 mM NaH.sub.2PO.sub.4, 0.5 M NaCl, 5 mM
Immidazole, pH 7.4) and mechanically lysed using an Avestin
homgenizer. The soluble fraction was separated by centrifugation
for 15 minutes at 23,300.times.g at 4.degree. C. The supernatant
was decanted and the pellet was solubilized in Lysis buffer (above)
supplemented with 4 M to 6 M guanidine hydrochloride (GdnHCl).
Solubilized protein was then purified on a suitably sized NiNTA
column (Qiagen, Inc.) pre-equilibrated with the GdnHCL supplemented
Lysis Buffer. The column was then washed with 5 to 10 column
volumes of the same buffer, followed by elution with the same
buffer supplemented with 300 mM Immidazole. The fractions eluted
off the column containing the protein of interest were diluted to
2-3 mgs/mL protein and then combined with a 1.2-1.5 molar excess of
solid NEM-PEG (40 kDa branched or other). The mixture was allowed
to react at room temperature for 30 minutes or until the reaction
was complete. The entire reaction volume was then placed into a
dialysis bag (5,000 Da Molecular Weight cutoff) and the mixture was
subjected to a dialysis refolding process. For example, this
process may consist of two 10-16 hour 500:1 (buffer:dialysate)
dialysis exchanges against 50 mM NaOAc, 150 mm NaCl, pH 4.5. The
dialysate from this procedure contains properly folded, PEGylated
materials plus excess reactants. The mixture of products and excess
reactants from the PEGylation reaction were clarified via
centrifugation or filtration prior to loading them onto a cation
exchange chromotography column (SP Sepharose or Resource S, GE
Healthcare). The column was developed with 150 mM to 1 M NaCl
gradient in the NaOAc background buffer. Studies to confirm the
pegylation were conducted as described above.
Example 4
Biophysical Characterization of .sup.10Fn3-Based Binders
Standard size exclusion chromatography (SEC) was performed on the
proteins purified from the HTPP and the midscale processes (0.1 to
1 .mu.g of protein for HTPP and 10-50 ug for midscale). SEC of HTPP
derived material was performed using a Superdex 200 5/150 column
(GE Healthcare) or on a Superdex 200 10/30 column (GE Healthcare)
for midscaled material on an Agilent 1100 or 1200 HPLC system with
UV detection at A.sub.214 nm and A.sub.280 nm and with fluorescence
detection (excitation=.sub.280 nm, emission=.sub.350 nm). A buffer
of 100 mM sodium sulfate, 100 mM sodium phosphate, 150 mM sodium
chloride, pH 6.8 at appropriate flow rate of the SEC column
employed. Gel filtration standards (Bio-Rad Laboratories, Hercules,
Calif.) were used for molecular weight calibration.
The results of the SEC on the HTPP purified .sup.10Fn3-based
binders showed predominantly monomeric proteins and elution in the
approximate range of 25 kDa vs. globular Gel Filtration standards
(BioRad).
The results of the SEC on the midscaled purified .sup.10Fn3-based
binders showed predominantly monomeric proteins and elution in the
approximate range of 25 kDa vs. globular Gel Filtration standards
(BioRad). FIG. 2 depicts exemplary SEC profiles for E/I
.sup.10Fn3-based binders (I1-GS10-E2 in FIG. 2A and E2-GS10-I1 in
FIG. 2B).
Select midscale .sup.10Fn3-based binders were further analyzed by
LC-MS (Water's 2695 liquid chromatography HPLC system coupled with
Waters Q-TOF API mass spectrometer, Waters Corporation, Milford,
Mass.). Samples were diluted to approximately 0.5 mg/ml with HPLC
grade water. Approximately 5 .mu.l of diluted sample was injected
onto a Jupiter C18 column (Catalog number 00G-4053-80, Phenomenex).
Buffer A: 0.02% TFA+0.08% formic acid in HPLC grade water. Buffer
B: 0.02% TFA+0.08% formic acid in HPLC grade acetonitrile. Sample
was eluted with gradient (Table 1) at flow rate 0.2 ml/minutes.
TABLE-US-00012 TABLE 1 Time % A B % 0 95 5 5.00 75 25 25.00 55 45
30.00 5 95 32.00 95 5 45.00 95 5
HPLC elution was split at approximately to 1:1 ratio and half sent
to UV detector and the other half to mass spectrometer. Mass
spectrometer had the following instrument settings: capillary
voltage 3.5 KV, cone voltage 40, source temperature 80.degree. C.,
desolvation temperature 250.degree. C., desolvation gas flow 450
and multi channel photo detector voltage 2200. Raw spectra were
deconvoluted with MaxEn1 (Waters Corporation).
The molecular weight of I1-GS10-E2 (SEQ ID NO: 22) as measured by
LC-MS is 24,445 Dalton, which is within 1 Dalton from the molecular
weight calculated from the amino acid composition. This indicates
that the protein has the correct amino acid composition and the N
terminal methionine is processed. There is no other post
translational modification on the protein.
Differential Scanning Calorimetry (DSC) analysis of the midscaled
I1-GS10-E2 was performed to determine the T.sub.m. A 1 mg/ml
solution was scanned in a N-DSC II calorimeter (Calorimetry
Sciences Corp) by ramping the temperature from 5.degree. C. to
95.degree. C. at a rate of 1 degree per minute under 3 atm
pressure. The data was analyzed versus a control run of the
appropriate buffer using a best fit using Orgin Software (OrginLab
Corp). The results of this assay demonstrate that the E/I binder
has a T.sub.m of 50.69.degree. C. (see FIG. 3A). Using the same
methods, the T.sub.m, of E2-GS10-I1 (with Peg) was determined to be
50.72.degree. C. and the T.sub.m of E2-GS10-I1 (without Peg) was
determined to be 56.82.degree. C. (see FIG. 3B).
Example 5
Determination of Binding Affinity
Surface plasmon resonance (BIAcore) analysis was performed on
solution-phase .sup.10Fn3-based binders in order to determine
off-rates and/or binding affinities using captured EGFR-Fc and
IGF1R-Fc. The extracellular domain of human IGF1R (aa 1-932) was
cloned into a mammalian expression vector containing the hinge and
constant regions of human IgG1. Transient transfection of the
plasmid produced a fusion protein, IGF1R-Fc which was subsequently
purified by Protein A chromatography. Recombinant human EGFR-Fc (aa
1-645 of the extracellular domain of human EGFR fused to human Fc)
was purchased from R&D systems (Minneapolis, Minn.). IGF1R-Fc
was captured on immobilized Protein A whereas EGFR-Fc was captured
on immobilized anti-human antibody.
In a typical experiment, anti-human IgG was immobilized on flow
cells 1 and 2 of a CM5 chip following the manufacturer's
recommendations (GE Healthcare, Piscataway, N.J.). EGFR-Fc (50 nM)
was injected at 5 uL for 2 minutes on flow cell 2 (Fc2). Two 30
second injections of 3 M MgCl.sub.2 were used for regeneration of
the bound EGFR-Fc from the anti-human IgG surface. Protein A was
diluted to 80 ug/mL in acetate pH 4.5 and immobilized to
.about.3000 RU on flow cells 3 and 4 of a CM5 chip surface.
Approximately 1300 RU of IGF1R-Fc was captured on Fc 4. Two 30
second injections of 50 mM glycine pH 1.5 were used to regenerate
the surface between samples.
A concentration series of 100 nM to 1 nM of HTPP purified protein
(three data points collected) or 300 nM to 0.05 nM of midscale
purified protein (eleven data points collected) was evaluated for
binding to EGFR-Fc or IGF1R-Fc. Sensorgrams were obtained at each
concentration and were evaluated using Biacore T100 Evaluation
Software, Version 1.1.1 (GE healthcare/Biacore) to determine the
rate constants k.sub.a (k.sub.on) and k.sub.d (k.sub.off). For the
HTPP evaluation the off-rate was fitted from the 3 point curves.
The affinity K.sub.D was calculated from the ratio of rate
constants k.sub.off/k.sub.on.
The EGFR .sup.10Fn3-based binders were evaluated for specificity in
a similar format using anti-human IgG to capture HER2-Fc. The
.sup.10Fn3-based binders did not show any discernible binding to
captured HER2-Fc under conditions where robust binding was seen for
EGFR-Fc.
As shown in Table 2, both domains of the E/I .sup.10Fn3-based
binders are functional, retaining their binding properties to the
respective targets. The off rates shown in Table 2 are from
midscale material and are similar to the qualitative results
obtained with the HTPP material.
TABLE-US-00013 TABLE 2 Summary of binding constants for
.sup.10Fn3-based binders Target Protein ka (1/Ms) kd (1/s) K.sub.D
(nM) EGFR-Fc E1 1.19E+05 1.18E-03 9.92 1.43E+05 1.89E-03 13.2
E1-GS10-I1 6.29E+04 4.74E-04 7.53 3.82E+04 3.89E-04 10.17
I1-GS10-E1 1.26E+05 6.03E-04 4.8 4.13E+04 4.25E-04 10.28 E2
3.73E+05 2.72E-04 0.73 3.27E+05 3.2E-04 0.98 E2-GS10-I1 3.93E+05
1.75E-04 0.45 3.75E+05 1.67E-04 0.45 I1-GS10-E2 6.47E+05 1.42E-04
0.22 3.90E+05 1.14E-04 0.29 E3 2.83E+05 3.98E-04 3.4 1.4 E3-GS10-I1
3.49E+05 2.29E-04 0.66 I1-GS10-E3 1.17E+05 2.91E-04 2.48 IGF1R-Fc
I1 3.84E+06 4.34E-04 0.11 E1-GS10-I1 5.13E+05 3.38E-04 0.66
I1-GS10-E1 1.47E+06 3.98E-04 0.27 E2-GS10-I1 1.24E+06 3.95E-04 0.32
I1-GS10-E2 3.82E+06 4.79E-04 0.13 E3-GS10-I1 1.8E+06 2.09E-04 0.12
I1-GS10-E3 1.37E+06 4.54E-05 0.03
Example 6
Inhibition of IGFR Activity in H292 Cells
The ability of E/I .sup.10Fn3-based binders to inhibit
phosphorylation of IGF1R on tyrosine 1131 was determined using an
H292 cell in vitro assay. Briefly, 65.times.10.sup.3H292 cells were
plated in 96-well microplates (Biocoat Poly-D-Lysine coated 96-well
plate, cat#356640, Becton Dickinson, Franklin Lakes, N.J.) in
RPMI-1640 culture medium containing 10 mM Hepes pH 7.4 and 10%
fetal bovine serum. Cells were allowed to adhere for 24 hours at
37.degree. C., 5% CO.sub.2. The next day cells were washed once
with 200 microliters per well of serum free RPMI-1640 and incubated
overnight in 100 .mu.L per well of serum free RPMI-1640. Serial
dilutions of HTPP material was added and cells were incubated for
an additional 3 hours. Cells were stimulated with 100 ng/ml of
IGF-1 (cat#500-P11, PeproTech, Rocky Hill, N.J.) for 10 minutes at
37.degree. C. Media was dumped from the plate and 100 .mu.L of cell
lysis buffer (Cell Signaling cat#9803, Beverly, Mass.) was added to
each well. Cells were incubated at room temperature for 15 minutes
to allow lysis and lysate was transferred to a phospho-IGFR ELISA
(cat#7302, Cell Signaling, Beverly, Mass.). The manufacturer's
procedure was followed to carry out the ELISA.
As demonstrated in FIG. 4, His tagged E1-GS10-I1 inhibited
IGF1-stimulated phosphorylation of the IGF1R (IC.sub.50=0.004 uM)
with comparable potency to the isolated IGF1R binder, I1
(IC.sub.50=0.018 uM). The EGFR binder, E1, alone had very little
effect on IGF1R phosphorylation (IC.sub.50>3.5 uM). As shown in
FIG. 9, additional E/I binders demonstrated ability to inhibit
IGF1R-stimulated phosphorylation with an IC50 in the range of 0.1
nM to 19 nM, including several pegylated E/I binders that were
tested. In particular, for the pegylated E/I binders E1-GS10-I1,
and I1-GS10-E1, inhibition of pIGFR was shown at 0.9 nM and 4 nM,
respectively. For pegylated E/I binders E2-GS10-I1 and I1-GS10-E2,
inhibition of pIGFR was shown at 0.3 nM and 0.8 nM,
respectively.
Example 7
Inhibition of EGFR Activity in H292 Cells
The ability of E/I .sup.10Fn3-based binders to inhibit
phosphorylation of the EGFR on tyrosine 1068 was determined using
an H292 cell in vitro assay. The assay was carried out as described
in Example 6, except that cells were stimulated with 100 ng/ml of
EGF (cat#236-EG-200, R & D Systems, Minneapolis, Minn.) and a
phospho-EGFR ELISA was performed (cat#7240, Cell Signaling,
Beverly, Mass.). The manufacturer's procedure was followed to carry
out the ELISA.
As demonstrated in FIG. 5, His-tagged E1-GS10-I1 inhibited
EGF-stimulated phosphorylation of the EGFR (IC.sub.50=0.020 uM)
with comparable potency to the isolated EGFR binder, E1
(IC.sub.50=0.007 uM). The IGF1R binder, I1 alone had very little
effect on EGFR phosphorylation (IC.sub.50>6.21 uM). As shown in
FIG. 9, additional E/I binders demonstrated ability to inhibit
EGF-stimulated phosphorylation with an IC50 in the range of 7 nM to
127 nM, including several pegylated E/I binders that were tested.
In particular, for pegylated E2-GS10-I1 and I1-GS10-E2, inhibition
of pEGFR was shown at 32 nM and 47 nM, respectively. Similar data
is shown in FIG. 11 for the pegylated E/I binders E2-GS10-I1, and
I1-GS10-E2.
Example 8
Inhibition of EGF+IGF1-Induced pAKT in H292 Cells
The ability of E/I .sup.10Fn3-based binders to inhibit
phosphorylation of AKT on serine 473 was determined using an H292
cell in vitro assay. The assay was carried out as described in
Example 6, except that cells were simultaneously stimulated with
both EGF and IGF1 as described above and lysates were analyzed with
a phospho-AKT ELISA (cat#7160, Cell Signaling, Beverly, Mass.). The
manufacturer's procedure was followed to carry out the ELISA.
Signal transduction at EGFR and IGF1R feeds into the PI3K-AKT
signaling pathway and stimulates phosphorylation of AKT. As
demonstrated in FIG. 6, E1-GS10-I1 inhibited EGF and
IGF1-stimulated phosphorylation of AKT in H292 cells. The E/I
.sup.10Fn3-based binder was slightly more potent in its ability to
block AKT activation (IC.sub.50=0.004 uM) than the IGF1R binder,
I1, by itself (IC.sub.50=0.031 uM). The EGFR binder, E1, exhibited
only modest activity in its ability to block AKT activation by both
ligands (IC.sub.50=1.28 uM). As shown in FIG. 9, additional E/I
binders demonstrated ability to inhibit EGF and IGF1-stimulated
phosphorylation of AKT with an IC50 in the range of 0.1 nM to 26
nM, including several pegylated E/I binders that were tested.
Example 9
Inhibition of Cell Proliferation in RH41 and H292 Cells
E/I .sup.10Fn3-based binders were evaluated for antiproliferative
activity in the H292 non-small cell lung carcinoma cell line, which
depends on EGFR signaling for growth, or the RH41 Ewing sarcoma
cell line, which depends on IGF1R signaling for growth.
Antiproliferative activity of binders was assessed in monolayer
cultures by staining cellular DNA with the CyQuantNF fluorescent
stain (cat#C35006, Invitrogen, Carlsbad, Calif.). Briefly,
2.times.10.sup.3H292 or 5.times.10.sup.3 RH41 cells were plated
into 96-well microplates (View Plates 96F cat#6005225,
Perkin-Elmer, Waltham, Mass.) in RPMI-1640 culture medium
containing 10 mM Hepes pH 7.4 and 10% fetal bovine serum and
allowed to adhere for 24 hours at 37.degree. C., 5% CO.sub.2. Cells
were maintained as exponentially growing monolayers and remained in
logarithmic growth phase during the period of the assay without
reaching confluence during the course of the assay. Twenty-four
hours after plating, serial dilutions of midscale material was
added and cells were incubated for an additional 72 hours.
Following this incubation, cells were treated with CyQuantNF
reagent and allowed to incorporate dye into cellular DNA for 1 hour
at 37.degree. C. Total DNA was quantified by reading fluorescence
at 485 nm excitation and 530 nm emission on a CytoFluor 4000
instrument (Applied Biosystems, Framingham, Mass.). Total time that
cells were exposed to drug was 72 hours. Standard compounds were
included in each experiment to verify assay performance and
reproducibility. Linear regression analysis of the percent of
inhibition by test compound was used to determine IC.sub.50
values.
As demonstrated in FIG. 7, in RH41 cells, His-tagged E1-GS10-I1
inhibited proliferation with comparable potency (IC.sub.50=0.009
uM) to the IGFR binder, I1 (IC.sub.50=0.028 uM). The EGFR binder,
E1, by itself had very little effect on the proliferation in this
cell line (IC.sub.50>12.5 uM).
As demonstrated in FIG. 8, in H292 cells, His-tagged E2-GS10-I1
inhibited proliferation with greater potency (IC.sub.50=0.329 uM)
than the IGFR binder, I1, (IC.sub.50=0.699 uM) or the EGFR binder,
E2 (IC.sub.50=0.553 uM). See Table 4 below for the IC50 values for
the E and I monomers.
Example 10
Competitive EGF Ligand Binding Assay
The E/I binders E1-GS10-I1, I1-GS10-E1, E2-GS10-I1 and I1-GS10-E2
(HTPP material) were tested in an EGF ligand binding cell-based
competition assay in A431 cells and compared to EGFR
.sup.10Fn3-based binders E1 and E2 (midscale material). A431 cells
were plated at 15000 cells/well in 96-well plates in DMEM+10% FBS
and incubated 48 hours. Cells were washed with starvation media
(DMEM+0.1% BSA) and incubated in starvation media for 1 hour.
Starvation media was removed and replaced with .sup.10Fn3-based
binders that were diluted in starvation media and cells were
pre-incubated for 30 minutes at 37.degree. C. to allow proteins to
bind to EGF receptors on cell surfaces. 10 nM final concentration
of Europium (Eu)-labeled EGF (Perkin Elmer, Boston, Mass.) diluted
in starvation media was added to pre-incubated cells and plates
were incubated for 3 hours at 4.degree. C. in the dark. Plates were
washed twice with cold PBS and 50 ul/well of Enhancement solution
(Perkin Elmer, Boston, Mass.) was added to plates and incubated 1
hour at 37.degree. C. Plates were read on the Flexstation II
(Molecular Devices). The data was plotted with Softmax plus
software and IC50 values, i.e., the concentration of
.sup.10Fn3-based binders required to inhibit 50% of the Eu-EGF
ligand from binding to the EGF receptor on the cell surfaces, were
calculated.
The results for E2 and E1 compared with E2-GS10-I1, I1-GS10-E2,
E1-GS10-I1 and I1-GS10-E1 are summarized in Table 3. This data
indicates that the E/I .sup.10Fn3-based binders compete with, and
inhibit the binding of, EGF to the EGFR receptor on A431 cells with
similar potency to the EGFR .sup.10Fn3-based binders. See Table 4
below for the IC50 values for the E and I monomers.
TABLE-US-00014 TABLE 3 Summary of IC50 values for inhibition of EGF
Binding to EGFR on A431 cell surfaces Protein IC50 (nM) E2 7 E1 14
E2-GS10-I1 1.8 I1-GS10-E2 1.4 E1-GS10-I1 14.6 I1-GS10-E1 7
Example 11
Activation and Signaling Activity in Cell-Based Assays
Target effects of the various E/I .sup.10Fn3-based binders were
evaluated in DiFi colon carcinoma cells by immunoblotting. Cells
were seeded at 4.times.10.sup.5 cells in each 25 cm.sup.2 flask and
incubated overnight at 37.degree. C. in 5% CO.sub.2. The next day,
treatments were initiated and cells were further incubated for
various times from 1.5 to 120 hours. Cells were then lysed in HNTG
(50 mM Hepes, 150 mM NaCl, 0.5% triton-X-100, 8% glycerol, 2 mM
Na.sub.3VO.sub.4, 1.5 mM MgCl.sub.2, 1 mM EDTA containing the
protease inhibitors AEBSF, aprotinin, leupeptin, bestatin,
pepstatin-A and E64) and total protein was quantified with the BCA
protein assay (Pierce, Waltham, Mass.). Levels of total EGFR, total
IGF1R and the phosphorylation state of the EGFR, MAP kinase protein
ERK1/2 isoforms, was detected by SDS-PAGE analysis of 20 micrograms
of total protein followed by transfer of proteins to nitrocellulose
and immunoblotting with specific antibodies. Blots were also probed
with .beta.-actin to demonstrate equal loading of each sample.
The pegylated E/I .sup.10Fn3-based binders E1-GS10-I1 (SEQ ID NO:
55), E2-GS10-I1 (SEQ ID NO: 56), and E3-GS10-I1 (SEQ ID NO: 53),
demonstrated the ability to degrade EGFR in this assay. In
addition, for E3-GS10-I1 (SEQ ID NO: 53), degradation of IGF1R was
also observed. The effect on EGFR degradation for the pegylated
binder E2-GS10-I1 is shown in FIG. 10, as are other effects on
signaling molecules. Additionally, the non-pegylated version of the
binder E2-GS10-I1 demonstrated similar EGFR degradation (data not
shown). FIG. 10 shows that for the pegylated binder I1-GS10-E2,
there was no EGFR degradation. Table 4 below summarizes various
properties of the E monomers.
TABLE-US-00015 TABLE 4 Summary of properties of E monomers. EGFR
Neutral- izes Inhibition Inhibition Inhibition BIAcore EGF of of of
H292 KD Binding pEGFR pERK Proliferation Monomer IC50 IC50 IC50
IC50 IC50 E1 14.6 nM 0.53 nM 18 nM 17 nM 18 nM E2 1.4 nM 1.46 nM 20
nM 40 nM 30 nM E3 0.72 nM 0.87 nM 11 nM 97 nM 26 nM
Example 12
Evaluation of Certain E/I .sup.10Fn3-Based Binders on H292 Tumor
Xenografts Grown in Nude Mice
The pegylated E/I binders E2-GS10-I1 and E3-GS10-I1 as well as the
monoclonal antibody panitumumab were evaluated in an H292 tumor
xenograft model. For in vivo models, panitumumab was obtained as
the marketed drug and E/I binders were purified as described above.
In vitro activity of all E/I binders was validated prior to
administration in animals by testing functionality of each end in
the EGF-stimulated pEGFR and the IGF1-stimulated pIGFR assay in
H292 cells. E/I binders were diluted in phosphate buffered saline
(PBS) at the beginning of the experiment and stored at 2-4.degree.
C. for the duration of each study. Both compounds were administered
i.p. in a total volume of 500 .mu.l/inj/mouse and were equilibrated
to room temperature prior to administration.
Mice and tumor propagation. Female athymic (nude) mice 5-6 weeks of
age were obtained from Harlan Sprague-Dawley Co. (Indianapolis,
Ind.). and were quarantined for approximately 3 weeks prior to
their use for tumor propagation or drug efficacy testing. The
animals were provided food and water ad libitum. Animal care was
performed in keeping with AAALAC and Bristol-Myers Squibb
guidelines. Tumors were propagated by subcutaneous (s.c.)
implantation in nude mice. Tumor passages occurred approximately
every two to four weeks.
In vivo antitumor testing. Estimated tumor weight was calculated
using the formula: Tumor weight (mg)=(w.sup.2*l)/2; where w=width
and l=length in mm Antitumor activity was evaluated in terms of %
tumor growth inhibition (TGI) where a % TGI of >50% was
considered active. Relative % tumor growth inhibition was
calculated as % TGI=[(C.sub.t-T.sub.t)/(C.sub.t-C.sub.0)].times.100
where C.sub.t=median tumor weight of control mice at time t in days
after tumor implant, T.sub.t=median tumor weight of treated mice at
time t, C.sub.0=median tumor weight of control mice at time 0. %
TGI value was calculated at various time points beginning after 1.5
tumor volume doubling times and sustained over a time period of 3
tumor volume doubling times (TVDT) where possible. Where,
TVDT=median time (days) for control tumors to reach target
size-median time (days) for control tumors to reach half the target
size. The definition of a cured mouse was one whose tumor was
undetectable, or <35 mg, when assessed more than 10 TVDTs
post-treatment. The dose of a compound which yielded the maximum
therapeutic effect, was termed the optimal dose (OD). Treatment
groups (typically 8 mice) with more than one death attributable to
drug toxicity were considered to have had excessively toxic
treatments and their data were not used in the evaluation of
antitumor activity. The maximum tolerated dose (MTD) is defined as
the dose level immediately below which excessive toxicity (i.e.
more than one death) occurred. Treated mice dying prior to having
their tumors reach target size were considered to have died from
drug toxicity. Statistical evaluations of data were performed using
Gehan's generalized Wilcoxon test (Gehan, E A, A Generalized
Wilcoxon Test for Comparing Arbitrarily Slightly-Censored Samples,
Biometrika 52:203-223, 1965).
Measurement of pharmacodynamic endpoints in tumors. Tumors were
harvested from untreated or drug treated mice and snap frozen in
liquid nitrogen. Samples were weighed and homogenized in 10 .mu.l
of lysis buffer (50 mM Hepes, 150 mM NaCl, 0.5% triton-X-100, 8%
glycerol, 2 mM Na.sub.3VO.sub.4, 1.5 mM MgCl.sub.2, 1 mM EDTA
containing one complete mini protease inhibitor tablet Sigma #S8820
per 15 ml buffer and phosphatase inhibitor cocktail Sigma #P5726)
for each mg of tissue. Tissues were minced in a 100 mm petri dish
with two scalpels, transferred to Falcon#2059 polypropylene round
bottom tubes and macerated with a hand held homogenizer for 30
seconds. Homogenate was transferred to 1.5 ml eppendorf tubes and
centrifuged at 15000.times.g for 2 minutes in a microfuge.
Clarified supernatant was transferred to a new tube and total
protein concentration was determined with the Pierce BCA protein
assay (Pierce Biotechnology). Samples were analyzed by
immunoblotting or on a Meso scale MSD Sector Imager 6000 multi spot
assay system as recommended by the manufacturer (Meso Scale
Discovery, Gaithersburg, Md.).
The pegylated E/I binders E2-GS10-I1 and E3-GS10-I1 were tested in
an H292 NSCLC in athymic mice. Tumors were implanted subcutaneously
with 1 mm.sup.3H292 tumor fragments in the hind flank and allowed
to establish to a size of 50-150 mg prior to initiation of
treatment on Day 6 post-tumor implant. The pegylated E/I binders
were administered i.p. at a dose of 100 mg/kg on a TIWX3 schedule
to assess antitumor activity. Panitumumab was obtained as marketed
drug and administered i.p. at its optimal dose of 1 mg/mouse and at
a lower dose of 0.1 mg/mouse on a Q3DX5 schedule. Mean tumor sizes
calculated from groups of 8 mice are shown in FIG. 12A. The 1
mg/mouse and 0.1 mg/mouse doses of panitumumab were both active by
% TGI with values of 101% and 100%, respectively and these values
were significantly different from control animals (p=0.0002, Table
5). Pegylated E2-GS10-I1 was also significantly active by % TGI
with a value of 96% (p=0.0005). Pegylated E3-GS10-I1 was not active
in this study with a % TGI value of 31% that was not statistically
different from the control group (p=0.416). Post dosing analysis
indicated that approximately two thirds of the pegylated E3-GS10-I1
was aggregated (66.64% aggregation/33.36% monomer for one batch and
72.53% aggregation/27.47% monomer for another batch) which could
account for the poor activity of pegylated E3-GS10-I1 in this
assay. In contrast, the pegylated E2-GS10-I1 showed only a small
percentage of aggregation in post dosing studies (1.79%
aggregation/98.21% monomer).
All treatments were well tolerated with no treatment related deaths
or excessive weight loss over the course of the study. Clinical
observations revealed no evidence of toxicity and the average
weight change over the course of therapy was within acceptable
limits (FIG. 12B).
TABLE-US-00016 TABLE 5 Results of the H292 human tumor xenograft
study Schedule, Dose AVE weight p value for Outcome Group Compound
Route (mg/kg) change (g) % TGI % TGI by % TGI 1 Control (untreated)
-- -- 5.3 -- 1.0 -- 2 panitumumab q3dx5; 6 1 mg/mse 9.6 101 0.0002
A ip.sup.a 3 panitumumab q3dx5; 6 0.1 mg/mse 6.3 100 0.0002 A
ip.sup.a 4 E2-GS10-I1 (w/ PEG) TIWx3; 6 100 -0.1 94 0.0005 A
ip.sup.a 5 E3-GS10-I1 (w/ PEG) TIWx3; 6 100 9.5 28 0.416 I ip.sup.a
.sup.aVehicle was phosphate buffered saline. Abbreviations used are
as follows: ip, intraperitoneal route; % TGI, relative % tumor
growth inhibition calculated as % TGI = [(Ct - Tt)/(Ct - C0)]
.times. 100 where Ct = median tumor weight of control mice at time
t in days after tumor implant, Tt = median tumor weight of treated
mice at time t, C0 = median tumor weight of control mice at time 0.
% TGI value was calculated at two points as the average inhibition
of Day 12 and Day 20. Outcome, a treatment regimen was considered
active if it produced a statistically significant % TGI value of
>50%; q3dx5; 6, compound was administered every three days for
five doses starting on the sixth day after tumor implant; TIWx3; 6,
compound was administered three times a week for three weeks
starting on the sixth day after tumor implant. p values were
calculated on Day 20 relative to the control group in a two tailed
paired analysis with 8 measurements per group. Outcome by % TGI, A
= active and I = inactive.
Pharmacodynamic endpoints from the H292 tumor study. Samples of
tumors from untreated control, panitumumab and E/I binder treated
groups were analyzed for levels of phosphorylated EGFR, ErbB2 and
IGFR that would indicate target suppression. Tumors were also
analyzed for levels of total EGFR to determine if EGF receptor
degradation occurred. On day 20, a final treatment was administered
and tumors were removed from 2 animals at 1 hour after dosing, 3
animals at 4 hours after dosing and 3 animals at 24 hours after
dosing. All treatments showed marked suppression of phosphorylated
EGFR and ErbB2 while the basal levels of phosphorylated IGFR were
too low to discern a difference in this study (FIG. 13). All
treatments showed a reduction in the amount of total EGFR
indicating degradation of the receptor had occurred.
Example 13
Selection and Characterization of MCF7 Cells Resistant to IGF1R
Inhibitor
MCF7 cells (American Type Culture Collection, Cat No. HTB-22,
Manassas, Va.) were cultured in RPMI medium containing 10 mM hepes
and 10% FBS at 37.degree. C. in the presence of 5% CO.sub.2. The
small molecule IGF1R inhibitor BMS-754807 was added to the culture
medium and the concentration increased at stepwise increments over
a period of 10 months until the cells exhibited continued
proliferation in the presence of 200 mM BMS-754807. The resistant
cells were designated MCF7r and the IC50 for BMS-754807 was 1239 nM
compared to 120 nM for the parental MCF7 cells as measured in a
proliferation assay carried out as previously described (Carboni et
al., Cancer Res. 69: 161-170 (2009)). The drug was then removed
from the culture medium and the MCF7r cells were passaged in
complete medium for an additional 20 or 60 days to remove all
traces of residual BMS-754807. Analysis of the MCF7r cells by
immunoblotting revealed that EGFR was significantly overexpressed
in the resistant cells compared to the parental MCF7 cells (FIG.
14). In addition, when MCF7 and MCF7r cells were serum starved and
then stimulated with EGF for 7 minutes, phosphorylated EGFR could
not be detected in the parental MCF7 cells (probably due to low
levels of EGFR) but was strongly visible in MCF7r cells. In serum
starved cells stimulated with IGF ligand, phosphorylated IGFR was
seen in the parental MCF7 cells but despite the slightly higher
levels of total IGFR present in the MCF7r cells almost no pIGFR was
observed. This shows that the IGFR in the resistant MCF7r cells
lost the ability to activate IGFR in response to IGF1 stimulation
(FIG. 14). Activation of the MAP kinase pathway in response to EGF
stimulation was stronger in the MCF7r cells as measured by pERK
activation.
Example 14
Antitumor Studies in MCF7 and MCF7r Xenografts
MCF7r cells were scaled up in T75 flasks and isolated by
centrifugation. Viable cell numbers were measured by trypan blue
exclusion with a Vi-CELL XR (Beckman Coulter, Fullerton, Calif.),
resuspended in PBS to 5.times.10.sup.6 viable cells/ml and
implanted subcutaneously in the hind flank of athymic mice in a
volume of 0.2 ml. For MCF7 and MCF7r tumor growth, all mice were
supplemented with 0.25 mg 90 day release pellets of
17-.beta.-estradiol (Innovative Research of America, Sarasota,
Fla., Cat. No. NE-121). Tumors were propagated until they reached a
median size of 500-1000 mg when they were excised and 1 mm.sup.3
fragments were reimplanted in the hind flank of new athymic mice.
Tumors were adapted for solid tumor growth by serial trocar passage
in mice through at least four rounds of growth during which tumor
volume doubling time and take rate were monitored for each passage.
Growth characteristics were observed to determine if the xenografts
exhibited acceptable properties to serve as a reliable,
reproducible model. The MCF7r tumor type demonstrated an acceptable
take rate and doubling time and therefore satisfied the criteria
for use as a xenograft model. The MCF7 parental tumor model had
been previously established using the same techniques. For the MCF7
parental xenograft, 1 mm.sup.3 tumor fragments were implanted
subcutaneously in the hind flank and allowed to establish to a size
of 50-150 mg prior to initiation of treatment on Day 13 post-tumor
implant. Cetuximab was obtained as marketed drug and administered
i.p. at its optimal dose of 1 mg/mouse and at a lower dose of 0.1
mg/mouse on a Q3DX5 schedule (doses administered on Day 13, 16, 19,
22, 25). Mean tumor sizes calculated from groups of 8 mice are
shown in FIG. 15A. In the MCF7 xenograft model, neither the 1
mg/mouse or the 0.1 mg/mouse dose of cetuximab was active by % TGI
with values of -9% and 3.2%, respectively and the tumor sizes were
not statistically different from the control group (Table 6).
For the MCF7r resistant xenograft, 1 mm.sup.3 tumor fragments were
implanted subcutaneously in the hind flank and allowed to establish
to a size of 50-150 mg prior to initiation of treatment on Day 6
post-tumor implant. Cetuximab was obtained as marketed drug and
administered i.p. at its optimal dose of 1 mg/mouse and at a lower
dose of 0.1 mg/mouse on a Q3DX5 schedule (doses administered on Day
6, 9, 12, 15, 18). Mean tumor sizes calculated from groups of 8
mice are shown in FIG. 15B. In the MCF7r xenograft model, doses of
cetuximab were active by % TGI with values of 105% and 75%,
respectively. The high dose of cetuximab had a TGI value over 100%
which indicates that it caused tumor regression below the starting
size at the initiation of treatment. Both doses resulted in a
statistically significant difference in tumor size compared to the
control group (Table 7).
TABLE-US-00017 TABLE 6 Results of the MCF7 human breast carcinoma
tumor xenograft study. Schedule, Dose p value for Outcome Group
Compound Route (mg/mouse) % TGI % TGI by % TGI 1 Control
(untreated) -- -- -- 1.0 -- 2 cetuximab q3dx5; 13 ip.sup.a 1 mg/mse
-9 0.223 I 3 cetuximab q3dx5; 13 ip.sup.a 0.1 mg/mse 3.2 0.220 I
.sup.aVehicle was phosphate buffered saline. Abbreviations used are
as follows: ip, intraperitoneal route; % TGI, relative % tumor
growth inhibition calculated as % TGI = [(Ct - Tt)/(Ct - C0)]
.times. 100 where Ct = median tumor weight of control mice at time
t in days after tumor implant, Tt = median tumor weight of treated
mice at time t, C0 = median tumor weight of control mice at time 0.
% TGI value was calculated at three points as the average
inhibition of Day 20, 24 and Day 27. Outcome, a treatment regimen
was considered active if it produced a statistically significant %
TGI value of >50%; q3dx5; 13, compound was administered every
three days for six doses starting on the thirteenth day after tumor
implant. p values were calculated on Day 24 relative to the control
group in a two tailed paired analysis with 8 measurements per
group. Outcome by % TGI, A = active and I = inactive.
TABLE-US-00018 TABLE 7 Results of the MCF7r human breast carcinoma
tumor xenograft study. Schedule, Dose p value for Outcome Group
Compound Route (mg/kg) % TGI % TGI by % TGI 1 Control (untreated)
-- -- -- 1.0 -- 2 cetuximab q3dx5; 6 ip.sup.a 1 mg/mse 105 0.001 A
3 cetuximab q3dx5; 6 ip.sup.a 0.1 mg/mse 75 0.024 A See footnotes
to Table 6. p values were calculated on Day 19 relative to the
control group in a two tailed paired analysis with 8 measurements
per group.
Example 15
Antitumor Studies in GEO Xenografts
GEO tumors were established by implanting 1 mm.sup.3 tumor
fragments subcutaneously in the hind flank of athymic mice and
allowing them to reach a size of 50-150 mg prior to initiation of
treatment on Day 18 post-tumor implant. Cetuximab was administered
ip at 0.25 mg/mouse on a Q3DX5 schedule (doses administered on Day
18, 21, 24, 27, 30). The IGFR kinase inhibitor BMS-754807 was
administered at 25 mg/kg on a QDX21 schedule. Mean tumor sizes
calculated from groups of 8 mice are shown in FIG. 16. Cetuximab
was active at 0.25 mg/mouse with a % TGI value of 67%. BMS-754807
was active with a % TGI of 80% and the combination of the two was
considerably more active then either agent alone with a % TGI of
94% (Table 8). All treatment groups were statistically different
from the control group on Day 26 (Table 8).
TABLE-US-00019 TABLE 8 Results of the GEO human colon carcinoma
tumor xenograft study. Cetuximab Dose BMS-754807 Schedule, (mg/
Schedule, Dose p Outcome Group Route mouse) Route (mg/kg) % TGI
value by % TGI Synergy 1 Control -- -- -- -- -- -- -- (untreated) 2
q3dx5; 6 ip.sup.a 0.25 mg/ -- 80 A -- mse 3 -- -- qdx21; 18.sup.b
25 67 A -- 4 q3dx5; 6 ip.sup.a 0.25 mg/ qdx21; 18.sup.b 25 94 A YES
mse .sup.aVehicle for cetuximab was phosphate buffered saline.
Vehicle for BMS-754807 was 50% polyethylene glycol 400, 50% water.
Abbreviations used are as described in Table 6 and synergy is
defined as statistically significant activity that is better than
either agent in the combination demonstrated on its own. Outcome by
% TGI, A = active and I = inactive.
Example 16
Antitumor Studies in 11292 Xenografts
H292 cells were implanted subcutaneously in the hind flank of
athymic mice as 1 mm.sup.3 fragments and allowed to establish to a
size of 50-150 mg prior to initiation of treatment on Day 12
post-tumor implant. Cetuximab was administered ip at 0.1 mg/mouse
on a Q3DX5 schedule. MAB391 is an antibody to IGF1R(R&D
Systems, Minneapolis, Minn., Cat. No. MAB391) and was administered
at a dose of 40 mg/kg on a BIWX3 schedule. Mean tumor sizes
calculated from groups of 8 mice are shown in FIG. 17. Cetuximab
was active at 0.1 mg/mouse with a % TGI value of 95.1% and MAB391
was inactive at 40 mg/kg with a % TGI value of 10.5% (Table 9).
Mice dosed with the combination of cetuximab and MAB391 exhibited a
% TGI value of 109.2% indicating tumor regression in the
combination group (Table 9). After dosing ceased, tumors regrew in
the cetuximab treated group more rapidly than in the group treated
with the combination of cetuximab and MAB391 (FIG. 17).
TABLE-US-00020 TABLE 9 Results of the H292 human NSCLC tumor
xenograft study. Cetuximab Dose MAB391 Schedule, (mg/ Schedule,
Dose p Outcome Group Route mouse) Route (mg/kg) % TGI value by %
TGI Synergy 1 Control -- -- -- -- -- -- -- (untreated) 2 q3dx5; 12
ip.sup.a 0.1 mg/ -- 95.1 A -- mse 3 -- -- BIWx3; 12.sup.a 40 10.5 I
-- 4 q3dx5; 12 ip.sup.a 0.1 mg/ BIWx3; 12.sup.a 40 109.2 A YES mse
.sup.aVehicle for cetuximab and MAB391 was phosphate buffered
saline. Abbreviations used are as described in Table 6 and 8.
Example 17
Colony Formation Assay
To determine the effects of test compounds on the ability to
inhibit colony formation of H292 cells, 400 cells were seeded into
24-well plates (Becton-Dickinson, Franklin Lakes, N.J., Cat. No.
351143) in complete medium and allowed to adhere overnight. The
next day medium was removed and replaced with medium containing 2%
FBS. Test compound was diluted into medium containing 2% FBS and
added to cells in serial dilutions. Cells were incubated at
37.degree. C. for 14 days. After 14 days, media was discarded and
wells rinsed once with 2 ml PBS. Cells were stained with 0.5 ml
Coomassie Stain Solution (Bio-Rad, Hercules, Calif., Cat. No.
161-0436) for 20 min. The stain was aspirated and wells were washed
quickly with 1.times. Destain Solution Coomassie R-250 (Bio-Rad,
Cat. No. 161-0438). A final rinse with 1 ml water per well was
carried out and plates were inverted and allowed to dry. Colonies
consisting of (at least) 50 cells or larger were counted by eye
under low power magnification (10.times.-20.times.). All samples
were tested in triplicate and IC50 values were calculated from
linear regression of the percent inhibition of control.
Representative results for a PEGylated E/I binder are shown in FIG.
18 and IC50 values for various E/I .sup.10Fn3-based binders,
monospecific IGF1R .sup.10Fn3-based binder, and EGFR antibody is
shown in Table 10.
TABLE-US-00021 TABLE 10 IC50 values of various E/I .sup.10Fn3-based
binders, monospecific IGF1R .sup.10Fn3-based binder, and EGFR
antibody in the colony formation assay. SAMPLE IC50 (nM) E4-GS10-I1
(with Peg) 5 I1-GS10-E5 (with Peg) 1 I1-GS10-E4 6 E2-GS10-I1 (with
Peg) 560 I1 monomer (with Peg) 15,510 panitumumab 140
Example 18
Epitope Mapping Assay
An epitope mapping assay was developed utilizing commercially
available antibodies where the binding site on the EGFR
extracellular domain is roughly known according to various
literature reports. The antibodies used in this assay are listed in
Table 11 and FIG. 19A depicts how antibodies were localized to
approximate binding domains on EGFR. The assay is a variation of
the In Cell Western assay previously described and assesses the
ability of EGFR .sup.10Fn3-based binders preincubated with A431 or
other cells expressing EGFR to block binding of the detection
antibodies from the panel listed in Table 11. The assay was carried
out as follows: A431 cells in log phase growth were harvested by
trypsinization and seeded in a 96 well plate at 24,000 cells/well
in a total volume of 100 .mu.l/well. The next day, media was dumped
and the EGFR .sup.10Fn3-based binders diluted in cold DMEM base
media were added to the plate and allowed to bind for 1 hour at
4.degree. C. to prevent internalization of EGFR. After binding,
cells were washed with 0.2 ml PBS+0.1% Tween-20 and fixed for 20
minutes in PBS+3.7% formaldehyde at room temp. Cells were blocked
in 0.2 ml of Odyssey blocking buffer for 1 hour at room temp. Next,
primary antibodies were diluted in 50 .mu.l of Odyssey blocker per
well and incubated for 1-2 hours at room temp. Primary antibodies
were dumped by inverting the plate, and each well washed 3.times.
with 200 .mu.l of PBS+0.1% Tween-20. Secondary antibodies are the
same ones used in the In Cell Western assay and were appropriate
for the species of antibody being detected. These secondary
antibodies were diluted (1:800) in Odyssey Blocker+0.2% Tween-20
and added in a volume of 50 .mu.l per well along with TOPRO3
(Invitrogen, Carlsbad, Calif., cat#T3605) diluted at (1:3000) to
counterstain cells for normalization. Cells were incubated on bench
for 1 hour at room temp. Secondary antibody was dumped out and each
well washed 4.times. with 200 .mu.l of PBS+0.1% Tween-20 for 5
minutes at room temp. Plates were imaged on a Licor instrument at
160 .mu.m resolution, medium quality, focus offset of 3 mm,
intensity of 5. This assay was also carried out with the marketed
drug antibodies cetuximab, panitumumab and nimotuzumab to determine
if the EGFR .sup.10Fn3-based binders were interfering with their
binding to EGFR on A431 cells. Representative results are shown in
FIG. 19B.
TABLE-US-00022 TABLE 11 Commercially available antibodies to the
extracellular domain of EGFR. Binding Clone SUPPLIER and cat#
SPECIES BINDS EPITOPE motif 1 Abcam ab38165 Rab h Peptide AA 42-58
linear 2 E234 Abcam ab32198 Rab h, mu, rat Peptide AA 40-80 (No
ICC) linear 3 N-20 Santa Cruz#31155 Goat IgG h AA 110-160 linear 4
ICR10 Abcam ab231 Rat IgG2a h(HN5) AA 124-176.sup.b,
neutra1izing.sup.e conf Santa Cruz#57095 5 EGFR1 Abcam ab30 Mu IgG1
h(A431) AA 176-294, neutralizing.sup.b conf Chemicon MAB88910 ab30
& MAB88910@(1 mg/ml) Labvision MS-311 6 199.12 Labvision
MS-396-P Mu IgG2a h AA 124-176, non- conf neutralizing.sup.b 7 LA22
Upstate 05-104 Mu IgG2a h(A431) AA 351-364, neutralizing.sup.a
linear 8 Abcam ab15669 Rab Mu, rat Peptide AA376-394.sup.d linear 9
225 Sigma E2156 Mu IgG1 h(A431) AA 294-475, neutralizing.sup.b,c
conf Labvision MS-269-P 10 528 Abcam ab3103 Mu IgG2a h(A431) AA
294-475, neutralizing.sup.b,c conf Santa Cruz#120 Labvision
MS-268-P 11 B1D8 Labvision MS-666-P Mu IgG2a h(A431) AA
294-475.sup.b conf 12 LA1 Upstate 05-101 Mu IgG1 h neutralizing 13
H11 Labvision MS-316-P Mu IgG1 h AA 294-475, non- linear
neutralizing.sup.b 14 111.6 Labvision MS-378-P Mu IgG1 h AA
294-475, neutralizing.sup.b linear Imgenex IMG80179 15 29.1 Sigma
E2760 Mu IgG1 h(A431) External carbohydrate non- Abcam ab10414
neutralizing Abbreviations: conf: epitope conformationally
specific; linear: epitope independent of conformation. .sup.aJBC
264(1989)17469 Ala351-Asp364, .sup.bJ Immunological Methods
287(2004)147, .sup.cMol Biol Med1(1983)511, .sup.dRaised against a
peptide to mouse EGFR [FKGDSFTRTPPLDPRELEI (SEQ ID NO: 491)],
.sup.eInt J Oncol 4(1994)277.
.sup.f[EEKKVCQGTSNKLTQLGTFEDHFLSLQRMFNNCEVVLGNLEITYVQRNYDLSFLKTIQEVAGYVLIA-
LNTVERIPLENLQIIRGNMYYENSYALAVLSNY (SEQ ID NO: 492)],
.sup.gIle-Gln-Cys-Ala-His-Tyr-Ile-Asp-Gly-Pro-His-Cys (SEQ ID NO:
493) (amino acids 580-591). .sup.hCancer Cell 7(2005)301.
Using various approaches, we have confirmed that the EGFR monomer
E3 binds to domain I of EGFR. Since other E monomers have similar
properties in various experiments, it is thought that the other E
monomers also bind to domain I of EGFR.
Example 19
Properties of I Monomers
BIAcore Analysis of the Soluble Fibronectin-Based Scaffold
Proteins
The kinetics of I monomers binding to the target was measured using
BIAcore 2000 or 3000 bio sensors (Pharmacia Bio sensor). A capture
assay was developed utilizing an IGF-IR-Fc fusion. A similar
reagent had been described by Forbes et al. (Forbes et al. 2002,
European J. Biochemistry, 269, 961-968). The extracellular domain
of human IGF-IR (aa 1-932) was cloned into a mammalian expression
vector containing the hinge and constant regions of human IgG1.
Transient transfection of the plasmid produced a fusion protein,
IGF-IR-Fc which was subsequently purified by Protein A
chromatography and captured on Protein A immobilized on Biasensor
CM5 chips by amine coupling. The kinetic analysis involved the
capture of IGF-IR-Fc on Protein A followed by injection of the
fibronectin-based scaffold protein in solution and regeneration of
the Protein A surface by glycine pH 2.0. Sensorgrams were obtained
at each concentration and were evaluated using a program
Biaevaluation, BIA Evaluation 2.0 (BIAcore), to determine the rate
constants k.sub.a (k.sub.m) and k.sub.d (k.sub.off) The
dissociation constant, K.sub.D was calculated from the ratio of
rate constants k.sub.off/k.sub.on. Typically, a concentration
series (2 uM to 0 uM) of purified fibronectin-based scaffold
protein was evaluated for binding to protein A captured human
IGF-IR-Fc fusion protein.
For experiments determining binding to human insulin receptor,
recombinant human insulin receptor (IR) and recombinant human
VEGF-R2-Fc were directly coupled to a CM5 Biasensor chip by amine
group linkage following standard procedures recommended by Biacore
(Uppsala, Sweden). In brief, 60 ug/mL of IR diluted in acetate 4.5
was coupled/immobilized to a level of 8300 RU and 11.9 ug/mL of
VEGF-R2-Fc diluted in acetate 5.0 was immobilized to a level of
9700 RU on flow cells 2 and 3. A blank reference surface was
prepared on FC1. Specific binding to either IR or VEGF-R2-Fc was
calculated by subtracting the binding observed to the blank
reference flow cell 1. Fibronectin-based scaffold proteins were
diluted to 10 uM in HBS-EP (10 mM Hepes, 150 mM NaCl, 3 mM EDTA,
0.05% Surfactant P20) and injected at 20 uL/min for 3 minutes over
the flow cells at 25.degree. C. and dissociation was observed over
10 mins
Cell-Based Receptor Blocking Assay
The human breast adenocarcinoma MCF-7 (ATCC, Manassas, Va.) was
plated in 24 well plates at a concentration of 50,000 cells per
well in RPMI 1640 (Invitrogen, Carlsbad, Calif.) containing 10%
fetal bovine serum (Hyclone, Logan, Utah). The following day, cells
were washed in binding buffer consisting of RPMI 1640 containing
0.1% BSA (Sigma, St. Louis, Mo.), and then pre-incubated for 30
minutes on ice in 200 .mu.L binding buffer containing IGF-IR
competitor. After the pre-incubation period, 40 pM
[.sup.125I]-IGF-I (Perkin Elmer, Wellesley, Mass.), equivalent to
approximately 60000 counts per minute, was added to each well and
allowed to incubate for an additional three hours on ice with
gentle agitation. The wells were then washed with ice cold PBS
containing 0.1% BSA. Cells were lysed with 500 .mu.L buffer
consisting of 0.1% SDS+0.5 N NaOH. Radioactivity of the lysates was
measured using a Wallac 1470 Gamma Counter (Perkin Elmer,
Wellesley, Mass.), and the data were analyzed using SigmaPlot
(Systat Software, Point Richmond, Calif.).
pIGFR Assay
Fibronectin-based scaffold proteins fused to Fc were evaluated for
their ability to inhibit IGF-1R phosphorylation in Rh41 human
rhabdomyosarcoma cells. A Western Blot was employed to assess the
ability of the I monomer to inhibit IGF-1R phosphorylation in Rh41
human rhabdomyo sarcoma cells. Cells were stimulated with IGF-I,
IGF-II, insulin ligands (50 ng/ml), or no stimulation (NS) and then
treated with various concentrations of the I monomer. Membranes
were probed with phospho-specific antibodies.
Cellular Proliferation in Rh41 (Human Rhabdomyosarcoma and H929
Human Multiple Myeloma)
Proliferation was evaluated by incorporation of [.sup.3H]-thymidine
into DNA after a 72 hour exposure to reagents. Rh41 cells were
plated at a density of 3500 cells/well in 96-well microtiter plates
and 24 hours later they were exposed to a range of I monomer
concentrations. After 72 hours incubation at 37.degree. C., cells
were pulsed with 4 .mu.Ci/ml [.sup.3H] thymidine (Amersham
Pharmacia Biotech, UK) for 3 hours, trypsinized, harvested onto
UniFilter-96, GF/B plates (PerkinElmer, Boston, Mass.) and
scintillation was measured on a TopCount NXT (Packard, CT). Results
are expressed as an IC50, which is the drug concentration required
to inhibit cell proliferation by 50% to that of untreated control
cells Data represents the average of triplicate wells with standard
deviations shown.
Results of the characterization of the I monomer are shown below in
Table 12.
TABLE-US-00023 TABLE 12 Properties of I monomers. IGFR Inhibition
of Neutralizes Inhibition of RH41 BIAcore IGF Binding pIGFR
Proliferation Monomer KD IC50 IC50 IC50 IC50 I1 0.11 nM 8 nM 0.2 nM
28 nM
Example 20
Additional Characteristics of Monospecific and Bispecific EGFR and
IGF-IR .sup.10Fn3-Based Binders
.sup.10Fn3-based binders that bound either EGFR or IGF-IR were
identified using the biochemical selection technique of mRNA
display in which a protein is covalently attached to its coding
nucleic acid sequences. .sup.10Fn3-based proteins-mRNA fusion
populations that bound either IGF-IR or EGFR when the receptors
were presented at concentrations from 1 to 10 nM were cloned into
E. coli and expressed as .sup.10Fn3-based proteins. A subset of
target binders that blocked EGFR or IGF-IR signaling and had
suitable biophysical properties were identified (Table 13). These
initial clones were optimized for target binding affinity and
cellular potency with additional mRNA selection at increasingly
lower target concentrations and selection for lower dissociation
rate constants. IC.sub.50 values obtained during the selection
procedures ranged from 9 to 304 nM, illustrating the opportunity
for choosing molecules from a wide range of potency values for the
construction of bi-specific .sup.10Fn3-based binders. EGFR
.sup.10Fn3-based binders were tested by In-Cell Western screening
assays for the blockade of phosphorylation of EGFR and ERK, a
downstream signaling molecule of EGFR activation (methods similar
to Example 1). Analogous studies were performed on optimized IGF-IR
binders. Optimized EGFR-binding clones (E3, E1, and E2) inhibited
EGFR phosphorylation on Y1068 and downstream phosphorylation of ERK
on Y204 of p42/p44 in vitro with IC.sub.50 values ranging from 9 to
40 nM, potencies that were more than 100-fold higher than the
parental EGFR clone (Table 13, methods similar to Example 1).
I1 bound to IGF-IR with a K.sub.D value of 0.11 nM and inhibited
IGF-1-stimulated IGF-IR phosphorylation with an IC.sub.50 of 0.2 nM
(Table 13, methods similar to Example 6). The optimized IGF-IR and
EGFR single-domain .sup.10Fn3-based binders were >95% monomeric
based on size exclusion chromatography, had melting temperatures
>56.degree. C. (Table 13, methods similar to Example 4), and
exhibited minimal immunogenic potential as predicted from EpiMatrix
(<7 for five out of six loops), a matrix-based algorithm for
T-cell epitope mapping (De Groot A S, Moise L (2007) Prediction of
immunogenicity for therapeutic proteins: state of the art. Curr
Opin Drug Discovery Devel 10:332-340). The .sup.10Fn3-based binders
E1, E2, and E3 were selected for further development, and had EGFR
binding constants in the range of 0.7 to 10 nM as determined from
Biacore assay (Table 13, methods similar to Example 5).
EGFR-binding of these .sup.10Fn3-based binders was competitive for
EGF binding to EGFR (Table 13) as measured by a displacement assay
using Europium labeled EGF (methods similar to Example 10).
Similarly, IGF-I binding to IGF-IR was inhibited by I1 (Table 13,
methods similar to Example 19).
Biophysical Characterization of Bi-Specific .sup.10Fn3-based
binders. T.sub.m values of selected E/I .sup.10Fn3-based binders
ranged from 49-58.degree. C. and their SEC profiles indicated the
protein was >90% monomer (Table 14, methods similar to Example
4). Monospecific .sup.10Fn3-based binders and E/I .sup.10Fn3-based
binders showed comparable binding affinities, although T.sub.m
values decreased slightly when the single domain .sup.10Fn3-based
binders were linked together (Tables 13 and 14). To increase serum
half life for in vivo applications, E/I .sup.10Fn3-based binders
were PEGylated with a 40 kDa branched PEG (methods similar to
Example 3). PEGylation of E/I .sup.10Fn3-based binders resulted in
a 10- to 20-fold reduction of binding affinity relative to the
un-PEGylated constructs due to decreased association rate constants
but did not decrease T.sub.m. Furthermore, PEGylation did not
markedly reduce inhibition of EGFR/IGF-IR phosphorylation in cells.
The PEGylated E-I orientation (wherein the EGFR binder is at the N
terminus, and IGF1R is at the C terminus) exhibited slightly lower
IC.sub.50 values for the inhibition of EGFR and IGF-IR
phosphorylation by ELISA compared to the I-E orientation. While
minor differences in the K.sub.D values and biological activity
were found between PEGylated E-I orientation, vs the I-E
orientation, there were no consistent trends.
TABLE-US-00024 TABLE 13 Properties of Monospecific .sup.10Fn3-based
binders Relevant to the Construction of E/I .sup.10Fn3-based
binders. A431 A431 EGFR IGF-IR pEGFR pERK H292 H292 Competition
T.sub.m SEC KD KD IC.sub.50, IC.sub.50, pEGFR pIGF-IR EGFR/IGF-IR
Name .degree. C. Monomer % nM nM nM nM IC.sub.50, nM IC.sub.50, nM
IC.sub.50, nM E- 56 ND 42.5 2580 2370 1148 .+-. 21 ND 29 .+-. 12.73
parent E3 60 >80 3.4 NA 15 .+-. 8 11 .+-. 7 22 .+-. 1 >7000
4.75 .+-. 1.77 E1 64 >95 9.92 NA 24 .+-. 7 13 .+-. 3 9 .+-. 2
>3400 15.9 .+-. 2.97 E2 72 >95 0.7 NA 38 .+-. 15 40 .+-. 9 31
.+-. 1 >3400 9.4 .+-. 3.68 I- ND ND NA 1.8 ND ND ND ND 13**
parent I1 61.5 >95 >6210 0.11 NA NA NA 0.2 8** ND, not done;
NA, not applicable; SEC, size exclusion chromatography. *IC.sub.50
values for EGFR and ERK phosphorylation levles in A431 cells were
determined by In-Cell Western assay (ICW). Phosphorylation levels
of EGFR and IGF-IR in H292 cells were determined by Enzyme-linked
immunosorbent assay (ELISA). **Competition for IGF-IR binding.
Standard deviations are from 3-6 experiments.
TABLE-US-00025 TABLE 14 Properties of the E/I .sup.10Fn3-based
binders. IGF- H292 A431 IR H292 pIGF-IR pERK A431 EGF-EGFR T.sub.m
EGFR K.sub.D pEGFR IC.sub.50, IC.sub.50, pEGFR Competition Name
.degree. C. K.sub.D nM nM IC.sub.50, nM nM nM IC.sub.50, nM
IC.sub.50, nM E3-GS10-I1 52 0.7 0.1 7 6 12 14 25 .+-. 6.5 E3-GS10-
52.5 10.4 0.74 10 6 40 42 80.5 .+-. 12.02 I1-PEG E1-GS10-I1 48 3.8
0.8 30 1 51 36 51 E1-GS10- 49 57.9 2.4 123 4 295 297 396 .+-. 223
I1-PEG E2-GS10-I1 56 0.5 0.2 8 0.1 20 19 2.1 .+-. 0.57 E2-GS10-
57.5 10.1 1.17 32 0.3 78 77 56.5 .+-. 24.5 I1-PEG I1-GS10- 60 3.6
0.46 47 0.8 118 97 128 .+-. 4.95 E2-PEG T.sub.m measurements are
from thermal scanning flurometry. K.sub.D values are from Biacore
binding assays using recombinant EGFR or IGF-IR domains adsorbed on
the chip. In-Cell Western assays (ICW) were conducted to determine
the ability of EI-Tandems to inhibit the phosphorylation of EGFR or
ERK in A431 cells. Enzyme-linked immunosorbent assays (ELISA) were
used to determine the phosphorylation of EGFR or IGF-IR in H292
cells.
Example 21
Species Cross-Reactivity of E/I .sup.10Fn3-Based Binders
Pegylated E/I .sup.10Fn3-based binders were analyzed for their
binding affinities to EGFR from mouse, rat and monkey using surface
plasmon resonance (BIAcore) analysis (methods identical to Example
5). Mouse EGFR was purchased from R&D systems (Minneapolis,
Minn.), rat EGFR was produced in house, and monkey EGFR was
purchased from KEMP (Frederick, Md.)
As shown in Table 15, all pegylated E/I .sup.10Fn3-based binders
bound to mouse, rat and monkey EGFR with low nanomolar affinities
indicating that all pegylated E/I binders are cross-reactive with
human, mouse, rat and monkey EGFR.
TABLE-US-00026 TABLE 15 KD (nM) KD (nM) KD (nM) Analyte (mouse
EGFR) (rat EGFR) (monkey EGFR) I1-GS10-E105 2.7 2.9 4.4 (pegylated)
I1-GS10-E5 3.4 3.6 5.1 (pegylated) I1-GS10-E4 5.5 3.7 3.9
(pegylated) E4-GS10-I1 6.9 5.6 5.7 (pegylated) E2-GS10-I1 9.6 9.6
18.0 (pegylated) I1-GS10-E85 13.9 10.7 7.0 (pegylated)
Example 22
Characterization of Additional E/I .sup.10Fn3-Based Binders
FIG. 43 summarizes various characteristics of additional E/I
.sup.10Fn3-based binders.
The pegylated E/I .sup.10Fn3-based binders were tested to determine
inhibition of EGF induced EGFR and ERK phosphorylation in A431,
using methods as previously described in Example 1. Results
demonstrated that the pegylated E/I .sup.10Fn3-based binders
inhibited EGF induced EGFR phosphorylation with IC50's ranging from
12 nM-297 nM and phosphorylation of ERK with IC50's ranging from 12
nM-295 nM (FIG. 43, columns a and b).
The ability of the pegylated E/I .sup.10Fn3-based binders to
inhibit IGFR and EGFR activity was also examined in H292 cells
using methods previously described in Examples 6 and 7. Results
indicated that the pegylated E/I .sup.10Fn3-based binders inhibited
IGFR activity with IC50's ranging from 0.2 nM-6 nM (FIG. 43, column
d) and inhibited EGFR activity with IC50's ranging from 1.3 nM-123
nM (FIG. 43, columns c).
The pegylated E/I .sup.10Fn3-based binders were tested to determine
if they could induce degradation of EGFR and IGFR in Difi cells as
shown in columns e and f of FIG. 43. Cells were treated with luM of
pegylated E/I .sup.10Fn3-based binders and harvested at time points
starting at 7 hrs and ending at 120 hrs and levels of EGFR and
IGF1R were determine by Western blot analysis. The strength of
degradation was scored as either (+) indicating the tandem degraded
that receptor but the degrdation was not sustained and receptor
expression reappeared during the time course or (++) which
indicates the tandem degraded the receptor and sustained that
degradation throughout the time course. Results (FIG. 43, column e
and f) demonstrated that the pegylated E/I .sup.10Fn3-based binders
displayed various patterns of EGFR and IGF1R degradation;
degradation of only IGFR, degradation of both EGFR and IGFR or no
degradation of either receptor. No tandem tested displayed the
ability to degrade only EGFR.
The binding affinity of the pegylated E/I .sup.10Fn3-based binders
for EGFR and IGF1R was assessed by surface Plasmon resonance
(BIAcore) analysis as previously described in Example 5. Results
demonstrated that the pegylated E/I .sup.10Fn3-based binders bound
to EGFR with affinities ranging between 3.35 nM-57.9 nM and bound
to IGF1R with affinities ranging between 0.37 nM-2.43 nM (FIG. 43,
columns g and h).
The pegylated E/I .sup.10Fn3-based binders were tested to determine
their potency for blocking EGF binding to EGFR on the surface of
A431 cells using methods previously described in Example 10. The
pegylated E/I .sup.10Fn3-based binders blocked EGF binding to A431
cells with IC50's ranging from 19.5 nM to 238 nM (FIG. 43, column
i).
The pegylated E/I .sup.10Fn3-based binders were assessed for their
ability to inhibit colony formation of H292 cells using methods
described in Example 17. As shown in FIG. 43, column j, the
pegylated E/I .sup.10Fn3-based binders inhibited colony formation
with IC50 values ranging from 1 nM-560 nM and three of the four
pegylated E/I .sup.10Fn3-based binders tested were 23-140 fold more
potent than the anti-EGFR monoclonal antibody panitumumab. The
fourth pegylated E/I .sup.10Fn3-based binders was 4 fold less
potent than panitumuab. The pegylated I1 monomer was only
marginally active in inhibiting colony formation in H292 with an
IC50>15 uM and this is expected since H292 cell growth is
predominantly driven by EGFR signaling and not IGF1R signaling.
The melting temperature was assessed for pegylated E/I
.sup.10Fn3-based binders by DSC (as previously described in Example
4) or thermal dye melt methodology. For thermal dye melt
assessment, the pegylated E/I .sup.10Fn3-based binders were diluted
to 0.2 mg/mL in 50 mM NaAc buffer pH 4.5. Each sample was spiked
with 1 uL of the 200.times. Sypro Orange in DMSO buffer for a final
concentration of 0.5% dye. Each sample was loaded into the 96 well
tray and coated with 5 uL of silicone oil. The tray was spun down
at 1,000 RPM and loaded onto the Bio-Rad CFX96 system and the
following method was selected: 25.degree. C. for 10 minutes+Plate
Read 25.degree. C. to 95.degree. C. @ 0.5.degree. C. increments for
15 minutes+Plate Read. Data analysis was performed for the
inflection point with the CFX software. As shown in FIG. 43, column
k, all pegylated E/I .sup.10Fn3-based binders had similar Tm
measurements, ranging from 49-62.5 degrees celsius. Tm measurements
for the pegylated E/I .sup.10Fn3-based binders were independent of
concentration and remained consistent at all concentrations tested.
DSC analysis of an exemplary binder, I1-GS10-E5 pegylated, measured
with a scan range of 15-95.degree. C. at 1 mg/ml protein
concentration in PBS, resulted in a Tm measurement of 55.2.degree.
C. as shown in FIG. 20.
Size exclusion chromatography (SEC) was performed on the pegylated
E/I .sup.10Fn3-based binders as previously described in Example 4.
SEC analysis revealed that all of the pegylated E/I
.sup.10Fn3-based binders were >95% monomeric as shown in FIG. 43
(column 1 of Table).
Example 23
Biochemical and Biophysical Properties of E/I .sup.10Fn3-Based
Binder I1-GS10-E5 Pegylated with Selected Amino Acid Changes
I1-GS10-E5 pegylated was constructed without the 6HIS tag (SEQ ID
NO: 487) and also with various alterations to the linker region. In
addition, a global change was made to all the constructs wherein
the C-terminal tail of the first monomer had a single point change
of the aspartic acid to glutamic acid (D to an E). Several clones
were made with selected serine residues mutated to cysteines (S to
C) to provide for alternate PEGylation sites. The effect of these
changes on biochemical and biophysical properties of the molecule
were compared and are summarized in Table 16. Methods for measuring
inhibition of pEGFR are described in Example 7, pIGFR in Example 6,
pERK in Example 1, Tm in Example 4, EGFR and IGFR KD in Example 5.
Detailed analysis of the binding kinetics were also carried out on
these clones and are presented in Tables 17 and 18 (using methods
similar to those described in Example 5).
TABLE-US-00027 TABLE 16 pEGFR pIGFR pERK EGFR IGFR IC50 IC50 IC50
KD KD SEC % CLONE NAME (nM) (nM) (nM) Tm (.degree. C.) (nM) (nM)
mono I1-GS10-E5 pegylated 28 2.2 12 56 2.7 0.25 96 I1-GS10-E5 30
1.2 11 56.8 Sticky.sup.(9) 0.23 94.1 pegylated.sup.(1)
I1-GSGCGS8-E5.sup.(3) 19.8 1.4 8 54.8 4 0.29 95.2
I1-GS10-E5-GSGC.sup.(4) 28.7 1.2 19 55 1.4 0.25 92.7 I1
(S62C)-GS10-E5.sup.(5) 21 1.9 10 55.5 8.7 0.7 97.45 I1-GS10-E5
(S62C).sup.(6) 68.4 2.2 30 56 1.7 0.26 96.12 I1
(S91C)-GS10-E5.sup.(7) 22.7 6.2 15 52 17 7.16 95.98 I1-GS10-E5
(S91C).sup.(8) 22.6 2.1 29 50.5 17.9 0.28 93.39 .sup.(1)No His Tag
was used for this construct. .sup.(2)a global change was made to
all the alternative constructs of I1-GS10-E5 pegylated, wherein the
C-terminal tail of the first monomer had a single point change of
aspartic acid to glutamic acid (D to an E). .sup.(3)The I1 mononer
linked with GSGC (SEQ ID NO: 489) plus GS8 (SEQ ID NO: 494), to E5.
.sup.(4)I1 linked with GS10 to E5 with GSGC (SEQ ID NO: 489) at the
tail of E5. .sup.(5)I1 linked with GS10 to E5, wherein the I1 has a
single point change of serine to cysteine at position 62.
.sup.(6)I1 linked with GS10 to E5, wherein the E5 has a single
point change of serine to cysteine at position 62. .sup.(7)I1
linked with GS10 to E5, wherein the I1 has a single point change of
serine to cysteine at position 91. .sup.(8)I1 linked with GS10 to
E5, wherein the E5 has a single point change of serine to cysteine
at position 91. .sup.(9)This construct demonstrated non-specific
binding to the flow cell so an accurate determination of affinity
was not possible in this experiment.
TABLE-US-00028 TABLE 17 Biacore binding of altered I1-GS10-E5
Pegylated clones to EGFR645-Fc. Description ka (1/Ms) kd (1/s) Kd
(nm) .DELTA.ka (fold) .DELTA.kd (fold) .DELTA.Kd (fold) I1-GS10-E5
Pegylated 2.93 .+-. 0.67E+04 7.24 .+-. 3.14E-05 2.69 .+-. 1.53 --
-- -- I1-GS10-E5 pegylated 2.27E+04 1.49E-04 6.6 0.8 0.5 0.4
I1-GS10-E5 pegylated.sup.(1) Non-specific binding to reference cell
surface at higher analyte concentrations (600 nM, 200 nM prohibited
kinetic value determination) ALTERNATIVE CLONES.sup.(2)
I1-GSGCGS8-E5.sup.(3) 2.94E+04 1.18E-04 4.0 1.0 0.6 0.7
I1-GS10-E5-GSGC.sup.(4) 3.34E+04 4.52E-05 1.4 1.1 1.6 2.0
I1(S62C)-GS10-E5.sup.(5) 2.28E+04 1.99E-04 8.7 0.8 0.4 0.3
I1-GS10-E5(S62C).sup.(6) 1.78E+04 3.04E-05 1.7 0.6 2.4 1.6
I1(S91C)-GS10-E5.sup.(7) 1.96E+04 3.34E-04 17.0 0.7 0.2 0.2
I1-GS10-E5(S91C).sup.(8) 1.08E+04 1.93E-04 17.9 0.4 0.4 0.2
.sup.(1)No His Tag was used for this construct. .sup.(2)a global
change was made to all the alternative constructs of I1-GS10-E5
pegylated, wherein the C-terminal tail of the first monomer had a
single point change of aspartic acid to glutamic acid (D to an E).
.sup.(3)The I1 mononer linked with GSGC (SEQ ID NO: 489) plus GS8
(SEQ ID NO: 494), to E5. .sup.(4)I1 linked with GS10 to E5 with
GSGC (SEQ ID NO: 489) at the tail of E5. .sup.(5)I1 linked with
GS10 to E5, wherein the I1 has a single point change of serine to
cysteine at position 62. .sup.(6)I1 linked with GS10 to E5, wherein
the E5 has a single point change of serine to cysteine at position
62. .sup.(7)I1 linked with GS10 to E5, wherein the I1 has a single
point change of serine to cysteine at position 91. .sup.(8)I1
linked with GS10 to E5, wherein the E5 has a single point change of
serine to cysteine at position 91.
TABLE-US-00029 TABLE 18 Biacore binding of altered I1-GS10-E5
Pegylated clones to IGF1R-Fc. Description ka (1/Ms) kd (1/s) Kd
(nm) .DELTA.ka (fold) .DELTA.kd (fold) .DELTA.Kd (fold) I1-GS10-E5
pegylated 1.04 .+-. 0.04E+06 2.62 .+-. 0.21E-04 0.25 .+-. 0.01 --
-- -- I1-GS10-E5 pegylated 1.10E+06 2.78E-04 0.25 1.1 0.9 1.0
I1-GS10-E5 pegylated.sup.(1) 1.28E+06, 2.88E-04, 0.22, 0.23 1.2 0.9
1.1 1.22E+06 2.76E-04 ALTERNATIVE CLONES.sup.(2)
I1-GSGCGS8-E5.sup.(3) 8.52E+05 2.45E-04 0.29 0.8 1.1 0.9
I1-GS10-E5-GSGC.sup.(4) 1.07E+06 2.65E-04 0.25 1.0 1.0 1.0
I1(S62C)-GS10-E5.sup.(5) 3.34E+05 2.34E-04 0.70 0.3 1.1 0.4
I1-GS10-E5(S62C).sup.(6) 1.07E+06 2.79E-04 0.26 1.0 0.9 1.0
I1(S91C)-GS10-E5.sup.(7) 8.22E+04 5.89E-04 7.16 0.1 0.4 0.04
I1-GS10-E5(S91C).sup.(8) 9.86E+05 2.81E-04 0.28 0.9 0.9 0.9
.sup.(1)No His Tag was used for this construct. .sup.(2)a global
change was made to all the alternative constructs of I1-GS10-E5
pegylated, wherein the C-terminal tail of the first monomer had a
single point change of aspartic acid to glutamic acid (D to an E).
.sup.(3)The I1 mononer linked with GSGC (SEQ ID NO: 489) plus GS8
(SEQ ID NO: 494), to E5. .sup.(4)I1 linked with GS10 to E5 with
GSGC (SEQ ID NO: 489) at the tail of E5. .sup.(5)I1 linked with
GS10 to E5, wherein the I1 has a single point change of serine to
cysteine at position 62. .sup.(6)I1 linked with GS10 to E5, wherein
the E5 has a single point change of serine to cysteine at position
62. .sup.(7)I1 linked with GS10 to E5, wherein the I1 has a single
point change of serine to cysteine at position 91. .sup.(8)I1
linked with GS10 to E5, wherein the E5 has a single point change of
serine to cysteine at position 91.
Example 24
Inhibition of Shared Downstream Signaling Pathways of EGFR and
IGFR
Inhibition of downstream signaling pathways were analyzed with a
pAKT ELISA identical to those previously described in Example 8.
Results of this study demonstrate that I1-GS10-E5 pegylated is more
potent than I1 pegylated alone at blocking IGF1-stimulated AKT
activation in H292 cells. E5 pegylated, the EGFR monospecific
binder alone did not efficiently prevent activation of AKT by IGF1
stimulation (FIG. 21).
Example 25
Inhibition of Cell Proliferation by .sup.10Fn3-Based Binders and
Comparator Antibody
H292 and RH41 cell proliferation experiments were conducted as
described in Example 9. The EGFR monospecific .sup.10Fn3-based
binder E5-pegylated inhibited proliferation of H292 cells with an
IC50 value of 0.016 .mu.M. The IGFR monospecific .sup.10Fn3-based
binder I1-pegylated had an IC50 value of >8.4 .mu.M while the
E/I .sup.10Fn3-based binder I1-GS10-E5 pegylated was slightly more
potent with an IC50 value of 0.006 .mu.M (FIG. 22). The H292 cell
line is of lung cancer origin and sensitive to inhibition of IGFR
and EGFR ((Akashi Y, et al. (2008) Enhancement of the antitumor
activity of ionising radiation by nimotuzumab, a humanised
monoclonal antibody to the epidermal growth factor receptor, in
non-small cell lung cancer cell lines of differing epidermal growth
factor receptor status. Br. J. Cancer 98:749-755; and Buck E, et
al. (2008) Feedback mechanisms promote cooperativity for small
molecule inhibitors of epidermal and insulin-like growth factor
receptors. Cancer Res. 68:8322-8332.)) In contrast, only the
I1-GS10-E5 pegylated binder and the I1-pegylated binder inhibited
the proliferation of RH41 cells (IC50 values were 0.0002 and 0.0004
.mu.M, respectively, FIG. 23). This was expected, since RH41 is a
pediatric rhabdomyo sarcoma cell line that is known to be driven
predominantly by IGFR signaling ((Huang F, et al. (2009). The
mechanisms of differential sensitivity to an insulin-like growth
factor-1 receptor inhibitor (BMS-536924) and rationale for
combining with EGFR/HER2 inhibitors. Cancer Res. 69:161-170)) and
thus not sensitive to EGFR blockade.
Example 26
Inhibition of Receptor Activation and Downstream Signaling In Vitro
by Pegylated and Non-Pegylated .sup.10Fn3-Based Binders
In order to understand the dynamics of EGFR/IGFR signaling and its
inhibition by I1-GS10-E5 pegylated, DiFi, H292 or BxPC3 cells were
serum-starved, exposed to 1 .mu.M or 0.1 .mu.M E5 pegylated, I1
pegylated, or I1-GS10-E5 pegylated, or vehicle control for 2 hours,
then stimulated with either EGF, IGF-I, or EGF+IGF-I for 10
min.
Cells were cultured in vitro, serum starved overnight and then
exposed to .sup.10Fn3-based binders for 2 hours prior to
stimulation with 100 ng/ml of EGF or IGF. Cell lysates were
prepared in lysis buffer (1% Triton X-100, 5% glycerol, 0.15 M
NaCl, 20 mM Tris-HCl pH 7.6, Complete Protease Inhibitor Cocktail
Tablets [Roche, Indianapolis, Ind.] and Phosphatase Inhibitor
Cocktail 2 [Sigma-Aldrich Corp.]). Lysates (30 .mu.g) were resolved
by SDS-PAGE, transferred to membranes, and immunoblotted with
antibodies to phospho-EGFR and total EGFR (Santa Cruz
Biotechnology, Carlsbad, Calif.), phospho-AKT (Ser 473),
phospho-p44/42 MAPK (Thr202/Tyr204) (Cell Signaling Technology,
Beverly, Mass.), or total actin (Chemicon International, Temecula,
Calif.) in Odyssey Blocking Buffer with 0.1% Tween 20 (LI-COR
Biosciences, Lincoln, Nebr.). Membranes were incubated with the
appropriate secondary antibodies. Protein visualization was
performed using a LI-COR Biosciences Odyssey infrared imaging
system.
As shown in FIG. 24, the basal levels of phosphorylated EGFR,
IGF-IR, and AKT were nearly undetectable after serum deprivation.
In DiFi cells, neither I1-GS10-E5 pegylated or E5 pegylated (mono
specific EGFR binder) are able to completely suppress
EGF-stimulated EGFR phosphorylation. In H292 and BxPC3 cells there
is strong inhibition of EGFR phosphorylation by both I1-GS10-E5
pegylated and E5 pegylated. In DiFi and BxPC3 cells, I1-GS10-E5
pegylated blocks IGF-stimulated IGFR phosphorylation more than I1
pegylated (mono specific IGFR binder) by itself. In H292 cells,
IGF-stimulation cross activates the EGFR only when EGFR is blocked.
I1-GS10-E5 pegylated inhibited EGF-stimulated pAKT in DiFi;
increased pAKT in EGF-stimulated H292 and in BxPC3 EGF did not
activate pAKT. In DiFi, H292 and BxPC3 cells I1-GS10-E5 pegylated
inhibited IGF-stimulated pIGFR more than the individual E5
pegylated and I1 pegylated by themselves. I1-GS10-E5 pegylated had
very little if any effect on EGF-stimulated pERK in DiFi, H292 or
BxPC3. IGF-stimulation did not induce pERK in any cell line
examined.
In another experiment with unPEGylated .sup.10Fn3-based binders,
H292 cells were serum-starved, exposed to 1 .mu.M unPEGylated
monospecific EGFR binder E2, IGFR binder I1, or E2-GS10-I1, or
vehicle control for 1 hour, then stimulated with either EGF, IGF-I,
or EGF+IGF-I for 10 min. The basal levels of phosphorylated EGFR,
IGFR, and AKT were nearly undetectable after serum deprivation
(FIG. 25). Stimulation with EGF induced EGFR phosphorylation, but
did not transactivate IGFR. EGFR phosphorylation was blocked by the
E2, and E2-GS10-I1, but not I1. Similarly, stimulation with IGF-I
induced strong phosphorylation of IGFR that was blocked by I1 and
E2-GS10-I1, but not by E2. EGF stimulation only slightly increased
AKT phosphorylation, but IGF-I or EGF+IGF-I strongly induced
phosphorylation of AKT that was suppressed to basal levels by both
I1 and E2-GS10-I1. The combination of IGF-I and EGF induced AKT
phosphorylation more than either growth factor alone. E2 partially
reduced pAKT induced by the combination of EGF and IGF-I. However,
I1 showed the most dramatic reduction in pAKT, suggesting that
stimulation with EGF+IGF-I led to strong AKT phosphorylation
through the IGFR pathway. Surprisingly, blockade of the EGFR
pathway by E2 followed by stimulation with EGF ligand actually
increased the phosphorylation of AKT, perhaps as a result of
EGFR-independent activation of AKT ((Dobashi Y, et al. (2009)
EGFR-dependent and independent activation of Akt/mTOR cascade in
bone and soft tissue tumors. Mod Pathol (Epub Ahead of Print)).
These results illustrate the complex cross-talk between the EGFR
and IGFR pathways and feed-back mechanisms.
Example 27
Competition Binding Studies with E/I .sup.10Fn3-Based Binders
For Biacore competition experiments, EGFR-Fc (3 .mu.g/mL in
Na-acetate pH 5.0) was immobilized on the Biacore CM5 chip surface
using standard EDC/NHS amide coupling chemistry to a surface
density of 300 RU. EGFR antibodies were obtained as a marketed drug
and competition between monospecific EGFR binder E2 and antibodies
for binding to EGFR-Fc was assessed by binding 450 nM E2 (30
.mu.L/min, 200 s contact time), immediately followed by 450 nM E2
alone, or a mixture of 450 nM E2 plus 450 nM cetuximab,
panitumumab, or nimotuzumab (30 .mu.L/min, 200 sec contact time).
The surface was successfully regenerated between cycles using two
10 sec pulses of 50 mM NaOH at a flow rate of 30 .mu.L/min Initial
injection of E2 shows binding to EGFR on the surface of the chip. A
second injection of E2 mixed with an equal amount of cetuximab,
panitumumab, or nimotuzumab shows no competition for binding of
antibodies to EGFR by E2 (FIG. 26A).
Surface plasmon resonance (BIAcore) analysis was utilized to
demonstrate simultaneous engagement of captured EGFR-Fc and
solution phase IGF1R by E/I .sup.10Fn3-based binders. Recombinant
human EGFR-Fc (aa 1-645 of the extracellular domain of human EGFR
fused to human Fc) was purchased from R&D systems (Minneapolis,
Minn.). Recombinant IGF1R (aa 1-932 of human IGF1R propeptide,
proteolytically cleaved and disulfide linked) was purchased from
R&D systems (Minneapolis, Minn.). To demonstrate simultaneous
engagement, anti-human IgG was immobilized on flow cells 1 and 2 of
a CM5 chip following the manufacturer's recommendations (GE
Healthcare, Piscataway, N.J.). EGFR-Fc (50 nM) was captured on flow
cell 2 at 10 uL/min for 2 minutes. Binding of E/I .sup.10Fn3-based
binders to EGFR-Fc was achieved by injecting .sup.10Fn3-based
protein samples (100 nM) over both flow cells at 10 uL/min for 2
minutes. Simultaneous engagement of EGFR-Fc and IGF1R was probed by
subsequently injecting IGF1R (0,100 nM) over both flow cells at 30
uL/min for 2 minutes. Dissociation of the complex was monitored for
300 seconds. Two 30 second injections of 3 M MgCl.sub.2 were used
for regeneration of the bound complex from the anti-human IgG
surface. Biacore T100 Evaluation Software, Version 2.0.1 (GE
healthcare/Biacore) was utilized to overlay sensograms and remove
airspikes. As shown in FIG. 26B, both domains of the E/I
.sup.10Fn3-based binder are functional and able to bind to EGFR-Fc
and IGF1R simultaneously.
Binding specificity of E2-GS10-I1 pegylated to HER family receptors
was assessed by Biacore as described in Example 5. HER-2-Fc,
HER-3-Fc and HER-4-Fc (R&D Systems) was captured on the surface
of the CM5 chip with anti-human IgG. E2-GS10-I1 pegylated did not
show any discernible binding to other HER family members under
conditions where robust binding was seen for EGFR-Fc (HER-1) (Table
19).
TABLE-US-00030 TABLE 19 Binding affinity of E2-GS10-I1 pegylated to
extracellular domains of HER family of receptors. EGFR-Fc HER-2-Fc
HER-3-Fc HER-4-Fc Name K.sub.D, nM* K.sub.D, nM K.sub.D, nM
K.sub.D, nM E2-GS10-I1 pegylated 10.1 >1000 >1000
>1000
Example 28
Measurement of Plasma Biomarkers
Levels of soluble biomarkers TGF.alpha. and mIGF1 were measured in
mouse plasma at the end of xenograft studies or in non tumor
bearing mice at various times following treatment. Blood was
obtained by terminal cardiac puncture into tubes containing EDTA as
an anticoagulant. Plasma was prepared by centrifuging blood at
1300.times.g for 10 minutes at 4 degrees C. and removing the
clarified supernatant to a separate tube. TGF.alpha. levels were
measured in 0.1 ml of plasma, mIGF1 levels were measured in 0.02 ml
plasma with an ELISA assay as recommended by manufacturer (R&D
Systems, Minneapolis, Minn.). Plasma levels of TGF.alpha. were
increased in mice treated with I1-GS10-E5 pegylated or the
monospecific EGFR binder E5 pegylated but not cetuximab (FIG.
27A-C). The TGF.alpha. could be secreted from the human tumor or
may represent endogenous mouse TGF.alpha.. Due to the high homology
between human and mouse TGF.alpha. (93% amino acid identity) the
ELISA may cross react with mouse TGF.alpha.. Furthermore, human
TGF.alpha. secreted by the implanted tumor can bind to the mouse
EGFR. Because I1-GS10-E5 pegylated and E5 pegylated can bind both
human and mouse EGFR, all host and tumor EGFR binding sites are
blocked by these .sup.10Fn3-based binders while cetuximab does not
bind mouse EGFR. To determine if these .sup.10Fn3-based binders
cause increases in endogenouse mouse TGF.alpha. and if the ELISA
cross reacts with mouse TGF.alpha., non-tumor bearing nude mice
were dosed with I1-GS10-E5 pegylated at 100 mg/kg and plasma
samples were taken at 4, 24, 48, 72 hours post dose. Increases in
mouse TGF.alpha. were in fact observed that persisted out past 72
hours (FIG. 28A). Plasma samples from non-tumored mice were also
tested for mIGF1 with a mouse specific ELISA and increases in this
ligand were also observed (FIG. 28B).
Example 29
Results of In Vivo Human Tumor Xenograft Studies for Various E/I
.sup.10Fn3-Based Binders
Several E/I .sup.10Fn3-based binders were evaluated in a
head-to-head H292 NSCLC study (methods described in Example 12) at
a lower dose than previously used so that differences in relative
activity could be ascertained. Efficacy of the E/I .sup.10Fn3-based
binders E2-GS10-I1 pegylated, E4-GS10-I1 pegylated, I1-GS10-E5
pegylated, I1-GS10-E85 pegylated, I1-GS10-E4 pegylated,
I1-GS10-E105 pegylated at a single dose of 0.625 mg/mouse and
panitumumab at two doses (1 mg/mouse and 0.1 mg/mouse) were
compared.
Both doses of panitumumab and all E/I .sup.10Fn3-based binders
evaluated in this study were active by a tumor growth inhibition
(TGI) endpoint. During the dosing phase, E4-GS10-I1 pegylated,
I1-GS10-E5 pegylated, I1-GS10-E4 pegylated and panitumumab all
caused tumor regression (Table 21, TGI values greater than 100%)
while E2-GS10-I1 pegylated, I1-GS10-E85 pegylated and I1-GS10-E105
pegylated caused tumor growth inhibition (Table 20, TGI values up
to 100%). Differences in activity were statistically significant
when compared to the control group. All treatments were well
tolerated with no treatment related deaths or excessive weight loss
over the course of the study. Comparison of the efficacy of the E/I
.sup.10Fn3-based binders and panitumumab are presented in Table 20
below and in FIG. 29. In FIG. 29A, measurements out to day 43 shows
the pattern of regrowth of the tumors after dosing ceased. FIG. 29B
shows measurements out to day 27 and the y-axis is expanded to
illustrate the relative differences in activity among the treatment
groups.
TABLE-US-00031 TABLE 20 In vivo antitumor activity in the H292
NSCLC study Schedule, Dose AVE weight p value for Outcome Group
Compound Route (mg/kg) change (g) % TGI % TGI by % TGI 1 Control --
-- 3.36 -- 1.0 -- (untreated) 2 panitumumab q3dx5; 6 ip.sup.a 1
mg/mse 5.19 107 0.0023 A 3 panitumumab q3dx5; 6 ip.sup.a 0.1 mg/mse
5.9 105 0.0029 A 4 E2-GS10-I1 TIWX3; 6 ip.sup.a 0.625 mg/mse -1.4
93 0.0067 A pegylated 5 E4-GS10-I1 TIWX3; 6 ip.sup.a 0.625 mg/mse
-0.23 105 0.0023 A pegylated 6 I1-GS10-E5 TIWX3; 6 ip.sup.a 0.625
mg/mse -2.92 103 0.0033 A pegylated 7 I1-GS10-E85 TIWX3; 6 ip.sup.a
0.625 mg/mse 1.08 86 0.0114 A pegylated 8 I1-GS10-E4 TIWX3; 6
ip.sup.a 0.625 mg/mse -1.21 103 0.0034 A pegylated 9 I1-GS10-E105
TIWX3; 6 ip.sup.a 0.625 mg/mse -1.54 95 0.0035 A pegylated
.sup.aVehicle was phosphate buffered saline. Abbreviations used are
as follows: ip, intraperitoneal route; % TGI, relative % tumor
growth inhibition calculated as % TGI = [(C.sub.t -
T.sub.t)/(C.sub.t - C.sub.0)] .times. 100 where C.sub.t = median
tumor weight of control mice at time t in days after tumor implant,
T.sub.t = median tumor weight of treated mice at time t, C.sub.0 =
median tumor weight of control mice at time 0. % TGI value was
calculated at two points as the average inhibition of Day 20, Day
24 and Day 27. Outcome, a treatment regimen was considered active
if it produced a statistically significant % TGI value of >50%;
q3dx5; 6, compound was administered every three days for six doses
starting on the sixth day after tumor implant; 6 on/1 off; 6,
compound was administered once a day for 6 days then no treatment
for 1 day and this regimen started on the sixth day after tumor
implant. p values were calculated on Day 20 relative to the control
group in a two tailed paired analysis with 8 measurements per
group.
Further in vivo studies were carried out with selected E/I
.sup.10Fn3-based binders below, in various xenograft models using
the methods described in Example 12. A description of the various
xenograft models is as follows: H292 is a non-small cell lung
carcinoma (NSCLC) and is described in more detail Example 12; MCF7r
breast carcinoma is described in Example 14; and GEO colon
carcinoma is described in Example 15. The DiFi human colon
carcinoma expresses high levels of activated EGFR and also
expresses IGFR; RH41 is a pediatric rhabdomyosarcoma cell line that
is known to be driven predominantly by IGFR signaling (Huang F, et
al. ((2009)) The mechanisms of differential sensitivity to an
insulin-like growth factor-1 receptor inhibitor (BMS-536924) and
rationale for combining with EGFR/HER2 inhibitors. Cancer Res.
69:161-170) and thus is not sensitive to EGFR blockade; Cal27 is a
human head and neck carcinoma expressing high levels of EGFR and
moderate levels of IGFR; BxPC3 is a human pancreatic carcinoma; and
H441 is a NSCLC.
Comparison of the efficacy of selected E/I .sup.10Fn3-based binders
are presented in Table 21. In these efficacy studies, all of the
E/I .sup.10Fn3-based binders showed equivalent activity to
panitumumab and all treatments were able to regress H292 tumors
below their starting size as indicated by % TGI values over 100%.
In the DiFi study, panitumumab regressed tumors at the 1 mg/mouse
dose and was active at the 0.1 mg/mouse dose while all of the E/I
.sup.10Fn3-based binders were inactive although the I1-GS10-E5
pegylated showed some inhibition of tumor growth (TGI=43.8%). In
the RH41 study, panitumumab was not active at either dose, the
E2-pegylated construct was not active while the E/I
.sup.10Fn3-based binders and the I1-pegylated construct were all
active. FIG. 32 shows antitumor efficacy in the RH41 model for a
representative construct E2-GS10-I1 pegylated (data also shown in
Table 22). In the Cal27 study panitumumab regressed tumors at the 1
mg/mouse dose and was active at the 0.1 mg/mouse dose but among the
E/I .sup.10Fn3-based binders only the I1-GS10-E5 pegylated E/I
.sup.10Fn3-construct was active.
Results of human tumor xenograft studies with I1-GS10-E5 pegylated
and individual I1 and E5 components designed to assess synergy are
presented in Table 22. These combination (synergy) studies were
structured such that the individual pieces of the E/I
.sup.10Fn3-based binders (ie., IGFR and EGFR monospecific pegylated
versions) were included so antitumor effects beyond the
contribution of isolated ends could be discerned. In the MCF7r
study, I1 pegylated was not active while the E5 pegylated, (E5
pegylated+I1 pegylated) and the I1-GS10-E5 pegylated clones were
all active and exhibited similar activity meaning that all of the
antitumor activity likely comes from inhibition of EGFR and
blocking the IGFR pathway did not provide any enhancement.
Cetuximab regressed tumors at the 1 mg/mouse dose and was not
active at the 0.1 mg/mouse dose. BMS-754807 was also not active
showing that blocking the IGFR pathway with a small molecule
inhibitor did not result in efficacy in this model.
In the BxPC3 study, I1 pegylated was not active while the E5
pegylated and (E5 pegylated+I1 pegylated) clones were active
(TGI=61.2% and 68.8%, respectively). The I1-GS10-E5 pegylated clone
was more active (TGI=78%) than the individual pieces it is made
from and the difference was statistically significant by a two
tailed paired t-test showing that it has synergistic activity in
this model. Cetuximab was active at all doses studied but adding in
IGFR inhibition by combining it with the I1-pegylated did not
result in synergy.
In the GEO study, I1 pegylated was not active while the E5
pegylated and (E5 pegylated+I1 pegylated) and I1-GS10-E5 pegylated
clones were active (TGI=83.5%, 92.1 and 92.1%, respectively). While
there may have been some enhancement provided by combining EGFR and
IGFR inhibition together in this model, the difference was not
significantly better than the E5 pegylated by itself. Cetuximab was
active at both doses studied but adding in IGFR inhibition by
combining it with the I1-pegylated did not result in synergy.
In the H441 study, I1 pegylated and E5 pegylated were not active on
their own but (E5 pegylated+I1 pegylated) was active (TGI=54.5%).
The I1-GS10-E5 pegylated clone was more active (TGI=69.2%) than the
individual pieces it is made from but the differences were not
statistically significant showing that it provides enhanced
activity but not synergy in this model. Cetuximab was active at the
1 mg/mouse dose and was not active at the 0.1 mg/mouse dose. Adding
in IGFR inhibition by combining it with the I1-pegylated did not
result in any enhancement in this model.
TABLE-US-00032 TABLE 21 In vivo results of selected E/I
.sup.10Fn3-based binders Dose AVE weight p value for Outcome Group
Compound Schedule (mg/kg).sup.a change (g) % TGI % TGI by % TGI In
vivo antitumor activity in the H292 study 1 Control (untreated) --
-- 5.7 -- 1.0 -- 2 panitumumab Q3dx5; 6 ip.sup.a 1 mg/mse 5.0 104
0.0006 A 3 panitumumab Q3dx5; 6 ip.sup.a 0.1 mg/mse 1.5 102 0.0005
A 4 E4-GS10-I1 pegylated TIWX3; 6 2 mg/mse -4.86 105 0.0004 A 5
I1-GS10-E5 pegylated TIWX3; 6 2 mg/mse -10.0 102 0.0006 A 6
I1-GS10-E4 pegylated TIWX3; 6 2 mg/mse -1.41 105 0.0005 A In vivo
antitumor activity in the DiFi study 1 Control (untreated) -- --
-0.5 -- 1.0 -- 2 panitumumab Q3dx5; 6 ip.sup.a 1 mg/mse 5.3 109.7
0.006 A 3 panitumumab Q3dx5; 6 ip.sup.a 0.1 mg/mse 2.6 99.9 0.005 A
4 E4-GS10-I1 pegylated TIWX3; 6 3 mg/mse -10.8 -1.1 0.815 I 5
I1-GS10-E5 pegylated TIWX3; 6 3 mg/mse -16.4 43.8 0.310 I 6
I1-GS10-E4 pegylated TIWX3; 6 3 mg/mse -8.5 1.4 0.977 I In vivo
antitumor activity in the RH41study 1 Control (untreated) -- -- 7.2
-- 1.0 -- 2 panitumumab q3dx5; 6 ip.sup.a 1 mg/mse 10.7 16.5 0.721
I 3 panitumumab q3dx5; 6 ip.sup.a 0.1 mg/mse 8.6 38.4 0.563 I 4
E4-GS10-I1 pegylated TIWX3; 6 2.5 mg/mse -5.3 72.7 0.02 A 5
I1-GS10-E5 pegylated TIWX3; 6 2.5 mg/mse -7.8 68 0.019 A 6
I1-GS10-E4 pegylated TIWX3; 6 2.5 mg/mse -2.9 64.5 0.018 A 7
Control (untreated) -- -- 12.3 -- 1.0 -- 8 E2-GS10-I1 pegylated
TIWX3; 18 2.5 mg/mse -1.8 58.6 0.044 A 9 E2-pegylated TIWX3; 18
1.25 mg/mse 5.9 20.2 0.530 I 10 I1 pegylated TIWX3; 18 1.25 mg/mse
7.1 58.6 0.025 A In vivo antitumor activity in the Cal27 study 1
Control (untreated) -- -- 9.4 -- 1.0 -- 2 Panitumumab q3dx5; 6
ip.sup.a 1 mg/mse 6.1 109.8 0.0006 A 3 panitumumab q3dx5; 6
ip.sup.a 0.1 mg/mse 5.8 72.9 0.003 A 4 E4-GS10-I1 pegylated TIWX3;
6 2 mg/mse -1.2 -11.4 0.587 I 5 I1-GS10-E5 pegylated TIWX3; 6 2
mg/mse -11.6 57.6 0.037 A 6 I1-GS10-E4 pegylated TIWX3; 6 2 mg/mse
-2.2 -9.2 0.177 I .sup.aVehicle was phosphate buffered saline for
all treatments. Abbreviations used are as follows: ip,
intraperitoneal route; po, oral route; % TGI, relative % tumor
growth inhibition calculated as % TGI = [(Ct - Tt)/(Ct - C0)]
.times. 100 where Ct = median tumor weight of control mice at time
t in days after tumor implant, Tt = median tumor weight of treated
mice at time t, C0 = median tumor weight of control mice at time 0.
% TGI value was calculated at two points as the average inhibition
on Day 19 and 23 for H292, Day 39 and 41 for DiFi, Day 34 and 37
for RH41 for groups 1-6 and Day 35, 36 and 39 for groups 7-10, Day
18 and 20 for Cal27. Outcome, a treatment regimen was considered
active if it produced a statistically significant % TGI value of
>50%; q3dx5; 6, compound was administered every three days for
six doses starting on the sixth day after tumor implant; TIWX3; 6,
compound was administered three times a week for 3 weeks and this
regimen started on the sixth day after tumor implant. p values were
calculated relative to the control group in a two tailed paired
analysis with 8 measurements per group on Day 23 for H292, Day 39
for DiFi, Day 37 for RH41 for groups 1-6 and Day 39 for groups 7-10
and Day 20 for Cal27.
TABLE-US-00033 TABLE 22 Summary of in vivo experiments with
.sup.10Fn3-based binders and comparators Dose AVE weight p value
for Outcome Group Compound Schedule (mg/kg).sup.a change (g) % TGI
% TGI by % TGI In vivo antitumor activity in the MCF7r study 1
Control (untreated) -- -- 6.1 -- 1.0 -- 2 I1 pegylated.sup.a TIWX3;
7 50 mg/kg, ip 17.1 -40.8 0.195 I 3 E5 pegylated.sup.a TIWX3; 7 50
mg/kg, ip 5.1 75.8 0.007 A 4 E5 pegylated.sup.a + I1
pegylated.sup.a TIWX3; 7 50 mg/kg, ip -3.0 81.8 <0.0001 A 5
I1-GS10-E5 pegylated.sup.a TIWX3; 7 100 mg/kg, ip -4.5 78 0.009 A 6
cetuximab.sup.a Q3DX5; 7 1 mg/mse, ip 11.7 105.4 0.0009 A 7
cetuximab.sup.a Q3DX5; 7 0.1 mg/mse, ip 7.5 34.3 0.031 I 8
BMS-754807.sup.b QDX14; 7 50 mg/kg, po -4.0 44.5 0.146 I In vivo
antitumor activity in the BxPC3 study 1 Control (untreated) -- --
3.1 -- 1.0 -- 2 I1 pegylated TIWX3; 9 50 mg/kg, ip 4.3 14.3 0.315 I
3 E5 pegylated TIWX3; 9 50 mg/kg, ip -5.3 61.2 0.0003 A 4 E5
pegylated + I1 pegylated TIWX3; 9 50 mg/kg, ip -4.9 68.8 0.0019 A 5
I1-GS10-E5 pegylated TIWX3; 9 100 mg/kg, ip -14.0 78.0 0.0002 A 6
cetuximab Q3DX5; 9 1 mg/mse, ip 5.2 62.6 0.0026 A 7 cetuximab
Q3DX5; 9 0.25 mg/mse, ip 2.5 62.8 0.0005 A 8 cetuximab + Q3DX5; 9 1
mg/mse, ip 3.6 62.1 0.0005 A I1 pegylated TIWX3; 9 50 mg/kg, ip In
vivo antitumor activity in the GEO study 1 Control (untreated) --
-- 7.5 -- 1.0 -- 2 I1 pegylated TIWX3; 9 50 mg/kg, ip -7.2 26.8
0.594 I 3 E5 pegylated TIWX3; 9 50 mg/kg, ip 9.7 83.5 0.0028 A 4 E5
pegylated + I1 pegylated TIWX3; 9 50 mg/kg, ip 5.4 92.1 0.0005 A 5
I1-GS10-E5 pegylated TIWX3; 9 100 mg/kg, ip -7.3 92.1 0.0006 A 6
cetuximab Q3DX5; 9 1 mg/mse, ip 7.7 91.8 0.0008 A 7 cetuximab
Q3DX5; 9 0.25 mg/mse, ip 7.8 92.0 0.0007 A 8 cetuximab + Q3DX5; 9 1
mg/mse, ip 7.1 91.3 0.0006 A I1 pegylated TIWX3; 9 50 mg/kg, ip In
vivo antitumor activity in the H441 study 1 Control (untreated) --
-- 12.4 -- 1.0 -- 2 I1 pegylated TIWX3; 9 50 mg/kg, ip 11.5 30.8
0.701 I 3 E5 pegylated TIWX3; 9 50 mg/kg, ip -8.8 43.1 0.292 I 4 E5
pegyalted + I1 pegylated TIWX3; 9 50 mg/kg, ip -0.8 54.5 0.011 A 5
I1-GS10-E5 pegylated TIWX3; 9 100 mg/kg, ip -3.9 69.2 0.022 A 6
cetuximab Q3DX5; 9 1 mg/mse, ip 12.6 65.2 0.002 A 7 cetuximab
Q3DX5; 9 0.25 mg/mse, ip 13.7 43.9 0.110 I 8 cetuximab + Q3DX5; 9 1
mg/mse, ip 10.2 66.7 0.060 I I1 pegylated TIWX3; 9 50 mg/kg, ip
.sup.aVehicle was phosphate buffered saline for all treatments.
Abbreviations used are as follows: ip, intraperitoneal route; po,
oral route; % TGI, relative % tumor growth inhibition calculated as
% TGI = [(Ct - Tt)/(Ct - C0)] .times. 100 where Ct = median tumor
weight of control mice at time t in days after tumor implant, Tt =
median tumor weight of treated mice at time t, C0 = median tumor
weight of control mice at time 0. % TGI value was calculated at two
points as the average inhibition on Day 22 and 26 for MCF7r, Day 23
and 27 for BxPC3, Day 29 and 31 for GEO and Day 17 and for H441.
Outcome, a treatment regimen was considered active if it produced a
statistically significant % TGI value of >50%; q3dx5; 6,
compound was administered every three days for six doses starting
on the sixth day after tumor implant; TIWX3; 6, compound was
administered three times a week for 3 weeks and this regimen
started on the sixth day after tumor implant. p values were
calculated relative to the control group in a two tailed paired
analysis with 8 measurements per group on Day 26 for MCF7r, Day 27
for BxPC3, Day 29 for GEO and Day17 for H441.
Example 30
Pharmacokinetic Profile of Various E/I .sup.10Fn3-Based Binders in
Mice
The pharmacokinetic profiles of the pegylated E/I .sup.10Fn3-based
binder, E2-GS10-I1, were assessed in mice via intraperitoneal
injection. Three nude mice per dose group were dosed with
E2-GS10-I1, formulated in PBS, at 10 and 100 mg/kg, ip and plasma
samples were collected in citrate phosphate dextrose solution at
pre dosing, 0.5, 2, 4, 8, 12, 24, 48, 72, 96, 144, and 168 hours
post dosing. Plasma samples were assessed for pegylated E2-GS10-I1
Fn3-based binder levels using a quantitative
electrochemiluminescence (ECL) assay developed to detect and
quantitate the pegylated E/I .sup.10Fn3-based binder in plasma
samples. In this assay, a mouse monoclonal antibody with
specificity toward the EGFR binding region was adsorbed to Meso
Scale Discovery plates overnight at 4.degree. C. to allow capture
of the pegylated E/I .sup.10Fn3-based binder in the plasma samples.
The plasma samples were added to the plates and incubated at
22.degree. C. for 1 h. The captured pegylated E/I .sup.10Fn3-based
binder was detected by a rabbit polyclonal antibody specific to the
scaffold region of the E/I .sup.10Fn3-based binder, mixed with a
goat anti-rabbit antibody linked with a SULFO-TAG. Following a wash
to remove unbound SULFO-TAG reagent, a read buffer was added and
ECL detection was used. The level of pegylated E2-GS10-I1 in plasma
samples was calculated based on comparison to a 4-parameter fit of
a standard curve of the pegylated E2-GS10-I1 Fn3-based binder.
Mice administered 10 or 100 mg/kg interperitoneally (ip) of
pegylated E2-GS10-I1 resulted in peak levels of approximately 200
and 1700 .mu.g/mL, respectively, indicating dose-proportional
pharmacokinetics (FIG. 30). Pharmacokinetic parameters for FIG. 30
were calculated in a similar fashion to those described in the
paragraph below (note that "T 1/2" is interchangeable with
"HL_lambda_z" and AUC is interchangeable with "AUCINF_obs". The
half-life of pegylated E2-GS10-I1 in mice was 15.75.+-.1.52 h (FIG.
30). Based on these pharmacokinetic parameters, administration of
100 mg/kg three times weekly (TIW) in human tumor xenograft studies
was able to maintain drug levels 10- to 100-fold higher than the in
vitro IC50 value.
Additional pharmacokinetic experiments were conducted on several
pegylated E/I .sup.10Fn3-based binders, where mice were
administered 10 or 100 mg/kg interperitoneally (ip) and, for the
pegylated I1-GS10-E5, 10 or 64 mg/kg sub-cutaneously (sc), plasma
was collected and analyzed as described above to measure the levels
of pegylated E/I .sup.10Fn3-based binders. The pharmacokinetic
parameters of these various E/I .sup.10Fn3-based binders were
obtained by non-compartmental analysis of plasma (serum)
concentration vs. time data. WinNonlin software (version 5.1,
Pharsight Corp. Mountain View Calif.) was used to calculate the
terminal half-life (HL_lambda_z), maximum observed concentration
(C.sub.max), the area under the curve from time zero extrapolated
to infinity (AUCINF_obs), clearance (CL_F_obs), volume of
distribution based on the terminal phase (Vz_F_obs) and the mean
residence time extrapolated to infinity (MRTINF_obs). Results
showed that the half life for the pegylated E/I .sup.10Fn3-based
binders were between 12.1-20.9 hours, as shown in FIG. 44 and FIG.
31.
Example 31
Pharmacodynamics
Samples were taken from the H292 and the DiFi xenograft models
described in Table 21 at the end of the study and processed as
outlined under Measurement of pharmacodynamic endpoints in tumors
in Example 12 for analysis of total levels of EGFR and IGFR protein
and phosphorylated EGFR and IGFR. Target effects of
I1-GS10-E5-pegylated and panitumumab were evaluated by
immunoblotting as described in Example 11. In FIG. 33A, levels of
total EGFR, pEGFR and total IGFR were lower in I1-GS10-E5-pegylated
treated tumors than in untreated tumors at the end of the DiFi
xenograft model. In FIG. 33B, levels of pEGFR were lower in tumors
treated with panitumumab and I1-GS10-E5-pegylated. Levels of total
EGFR were lower only in I1-GS10-E5-pegylated treated tumors but not
in panitumumab treated tumors. Levels of total IGFR were lower in
both I1-GS10-E5-pegylated treated tumors and in one panitumumab
treated tumor but not the other. The amount of pIGFR in these
models was too low to detect differences following treatment.
Immunoblots were probed with GAPDH to demonstrate equal loading of
protein.
Example 32
EGFR .sup.10Fn3-Based Binders Optimization and Consensus Sequence
Analysis
The .sup.10Fn3-based binder 679F09 (as described in PCT WO
2009/102421) (FIG. 34) was identified as a binder to EGFR
ectodomain-Fc fusion protein (R&D Systems). Binding activity
was selected using a bead coated with EGFR-Fc and .sup.10Fn3-based
binders coupled to their nucleic acid coding sequence (see e.g., Xu
et al., Directed Evolution of High-Affinity Antibody Mimics Using
mRNA Display, Chem. Biol. 9: 933-942 (2002)). More potent variants
of the parental EGFR binder 679F09 having alterations to the amino
acid sequences in the BC, DE and FG loops were also identified.
Sequence Analysis I: All .sup.10Fn3-Based Binders Selected for
High-Affinity Binding to EGFR
In order to reveal sequence patterns that defined strong affinity
for EGFR, all unique EGFR binding sequences (1044) were analyzed
using several methods. First, the sequences were analyzed by the
frequency of amino acids at each position in the loops (FIGS.
35-38). Only unique sequences for each loop were analyzed.
From the above sequence analysis, the following broad sequence
motif was defined:
Sequence Motif #1 (a) BC loop: "YQ" in positions 7-8 (i.e.,
corresponding to positions 29 and 30 of SEQ ID NO: 1) (b) DE loop:
aliphatic residue ("V/I/L/M/A") in position 3 (i.e., corresponding
to position 54 of SEQ ID NO: 1) (c) FG loop: "D/N" in position 1
(i.e., corresponding to position 77 of SEQ ID NO: 1)
All 1044 sequences analyzed, except one, follow the FG loop
sequence pattern (c). Of all unique sequences analyzed, 90% follow
pattern (a) for the BC loop, and 95% follow pattern (b) for the DE
loop. All sequences analyzed, except four, follow at least two of
the three patterns above. In addition, the 15-amino acid FG loop
length is a noteworthy sequence feature.
In addition to the broad Sequence Motif #1 defined above, the data
in FIGS. 35-38 were used to define a second sequence motif based on
the dominant residues at each position. Residues were included in
this motif if the sum of the top 3 most frequent amino acids had a
greater than 50% frequency.
Sequence Motif #2 (a) BC loop: XXXXXXYQ (same as Motif #1), wherein
X is any amino acid (b) DE loop: (G/Y/H)(D/M/G)(V/L/I)X, wherein X
is any amino acid (c) FG loop, 10 amino acid length:
(D/N)(Y/M)(Y/A/M)(Y/H/F)(K/Q/V)(E/P/R)(Y/T/K)X(E/Y/Q)(Y/G/H),
wherein X is any amino acid (d) FG loop, 15 amino acid length:
D(Y/F/W)(Y/F/K)(N/D/P)(P/H/L)(A/T/V)(T/D/S)(H/Y/G)(E/P/V)(Y/H)(T/K/I)(Y/F-
)(H/N/Q)(T/Q/E)(T/S/I)
The analysis methods used to define Sequence Motifs #1 and #2
evaluate each residue position within a loop separately. To reveal
any sequence motifs spanning multiple residues within a loop, the
.sup.10Fn3-based binders were subjected to further analysis. In
this analysis, the loop sequences were aligned using ClustalW
(Thompson J D et al. CLUSTAL W: improving the sensitivity of
progressive multiple sequence alignment through sequence weighting,
position-specific gap penalties and weight matrix choice. Nucleic
Acids Research 22: 4673-4680, 1994). From this alignment, families
of sequences were grouped using manual inspection. For the BC and
DE loops, sequence patterns similar to Sequence Motifs #1 and #2
were observed. However, additional sequence motifs could be defined
for the 10 and 15 amino acid long FG loops.
Sequence Motif #3 (a) FG loop, 10 amino acid length (1)
DY(A/Y)GKPYXEY (SEQ ID NO: 473), wherein X is any amino acid (2)
DY(A/Y)Y(K/R/Q/T)PYXEY (SEQ ID NO: 474), wherein X is any amino
acid (3) (D/N)Y(A/Y)(Y/F)(K/R/Q/T)EYXE(Y/H) (SEQ ID NO: 475),
wherein X is any amino acid (4) DYY(H/Y)X(R/K)X(E/T)YX (SEQ ID NO:
476), wherein X is any amino acid (5)
DYY(H/Y)(K/H/Q)(R/K)T(E/T)Y(G/P) (SEQ ID NO: 477) (6)
(D/N)MMHV(E/D)YXEY (SEQ ID NO: 478), wherein X is any amino acid
(7) DYMHXXYXEY (SEQ ID NO: 479) (like FG loop of 679F09), wherein X
is any amino acid (8) D(M/Y)YHX(K/R)X(V/I/L/M)YG (SEQ ID NO: 480),
wherein X is any amino acid (b) FG loop, 15 amino acid length (1)
D(Y/F)(Y/F)NPXTHEYXYXXX (SEQ ID NO: 481), wherein X is any amino
acid (2) D(Y/F)(Y/F)D(P/L)X(T/S)HXYXYXXX (SEQ ID NO: 482), wherein
X is any amino acid (3) D(Y/F)(K/R)PHXDGPH(T/I)YXE(S/Y) (SEQ ID NO:
483), wherein X is any amino acid
Sequence Analysis II: .sup.10Fn3-Based Binders Showing More Potent
Inhibition of EGFR Phosphorylation
Another overall sequence analysis was performed on the subset of
.sup.10Fn3-based binders that showed the most potent activity in a
cell-based assay (as opposed to Sequence Analysis I, which was
performed on all binders selected for high-affinity binding to EGFR
through Profusion). Because many of the binders were only run
through single-point cell-based assays, binders that showed greater
than 75% inhibition of EGFR phosphorylation at a fixed
concentration of 100 nM were included in this analysis. The percent
inhibition at a given concentration is related to the IC50 by: %
inhibition=100.times. concentration/(concentration+IC50).
Normally, an IC50 is calculated by fitting the data for %
inhibition at various concentrations. However, given that only a
single data point is available for each binder, it is inappropriate
to use this single data point to calculate an IC50. Therefore, the
percent inhibition of EGFR signaling at a single concentration
point was used as an approximation of the potency of the binder.
Although a binder may show 75% inhibition at a concentration of 100
nM, increasing the concentration will allow the clone to show 100%
inhibition at a higher concentration. The % inhibition is inversely
related to the IC50; i.e., the higher the % inhibition, the lower
the IC50 and the more potent the binder. If a binder showed 75%
inhibition at a concentration of 100 nM, we considered this to be a
"potent" binder for the purposes of Sequence Analysis II. However,
the binders which showed less than 75% inhibition at 100 nM
concentration for the most part still bind to EGFR and still have
an effect on EGFR signaling. For instance, the anti-EGFR monoclonal
antibody Nimotuzumab (Friedlander E et al. ErbB-directed
immunotherapy: antibodies in current practice and promising new
agents. Immunol Lett 116: 126-140, 2008) is currently under
development as a therapeutic, but it shows <5% inhibition at a
100 nM concentration in the EGFR phosphorylation assay (data not
shown). The sequences of all "potent" binders assayed and their %
inhibition of EGFR phosphorylation at 100 nM concentration is shown
in FIG. 45.
The total number of unique .sup.10Fn3-based binders that showed
>75% inhibition at 100 nM concentration was 111. As before, the
sequences first were analyzed by the frequency of amino acids at
each position in the loops (FIGS. 39-42). Since these binders are a
subset of all the binders selected for high affinity binding to
EGFR during Profusion, they also follow Sequence Motif #1 (see
above). All "potent" sequences analyzed follow the FG loop sequence
pattern ("D/N" in position 1). Of all unique "potent" sequences
analyzed, 93% follow the pattern for the BC loop ("YQ" in positions
7-8), and 98% follow the pattern for the DE loop (aliphatic residue
("V/I/L/M/A") in position 3). All "potent" sequences analyzed
follow at least two of the three patterns of Sequence Motif #1.
Of note, the 15-amino acid FG loop length also appears to be highly
represented in the most "potent" binders. While 15-amino acid long
FG loops represent only 55% of all binders selected for high
affinity binding to EGFR (Sequence Analysis I), 15-amino acid FG
loops represent 86% of the binders with >50% inhibition of EGFR
phosphorylation at 100 nM concentration, and 91% of the binders
with >75% inhibition ("potent" binders in Sequence Analysis II).
Therefore, the longer 15-amino acid FG loop appears to be a
sequence pattern associated with greater potency.
Of the 111 "potent" sequences analyzed, only 10 contain 10-amino
acid long FG loops, and 6 of those are unique. Therefore, a single
sequence motif can encompass every "potent" 10-amino acid FG loop
sequence. Sequence Motif #4 was defined based on these 6
sequences.
Sequence Motif #4 FG loop, 10-amino acid length, "potent" binders
(D/N)(M/Y)(M/A/W)(H/F/Y)(V/K)EY(A/Q/R/S/T)E(Y/H/D)
The sequence analysis of the "potent" binders with 15-amino acid FG
loops also further illuminated which residue positions were most
conserved, allowing Sequence Motif #5 to be defined. An "X" in this
sequence motif denotes positions where there are no three dominant
amino acids.
Sequence Motif #5 FG loop, 15-amino acid length, "potent" binders
D(Y/F/W)(Y/F/K)(N/P/D)(P/H/L)X(T/D/S)(H/G/Y)(E/P/Y)(Y/H)XYXXX,
wherein X is any amino acid
All of the EGFR binders that were analyzed are progeny of the
parent 679F09 and constitute a sequence "family," i.e. they are all
related in sequence according to the aforementioned sequence
motifs. Various members of the 679F09 family of binders can
tolerate a T51I scaffold mutation and retain binding activity.
Therefore, a T51I scaffold mutation could be combined with any of
the aforementioned sequence motifs to also yield a binder with high
affinity binding to EGFR.
Finally, it should be noted that amino acids with similar
properties can often be substituted into protein sequences with
little or no effect on structure or function. This indeed is the
case for .sup.10Fn3-based binders as well, where conservative amino
acid substitutions in either the loop or scaffold regions can still
lead to binders which bind to EGFR. For instance, substituting "Y"
for "H" in the second position of the FG loop of binder E98 yields
binder E99, and both binders show similar potency in inhibiting
EGFR phosphorylation (FIG. 45).
SEQUENCE LISTINGS
1
495194PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 1Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr Pro Thr1 5 10 15Ser Leu Leu Ile Ser Trp Asp Ala Pro Ala
Val Thr Val Arg Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly
Asn Ser Pro Val Gln Glu Phe 35 40 45Thr Val Pro Gly Ser Lys Ser Thr
Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr
Val Tyr Ala Val Thr Gly Arg Gly Asp65 70 75 80Ser Pro Ala Ser Ser
Lys Pro Ile Ser Ile Asn Tyr Arg Thr 85 90294PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
2Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr1 5
10 15Ser Leu Leu Ile Ser Trp Asp Ala Pro Ala Val Thr Val Arg Tyr
Tyr 20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln
Glu Phe 35 40 45Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly
Leu Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr
Gly Ser Gly Glu65 70 75 80Ser Pro Ala Ser Ser Lys Pro Ile Ser Ile
Asn Tyr Arg Thr 85 90390PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 3Val Ser Asp Val Pro Arg
Asp Leu Glu Val Val Ala Ala Thr Pro Thr1 5 10 15Ser Leu Leu Ile Ser
Trp Ser Ala Arg Leu Lys Val Ala Arg Tyr Tyr 20 25 30Arg Ile Thr Tyr
Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40 45Thr Val Pro
Lys Asn Val Tyr Thr Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly Val
Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Arg Phe Arg Asp65 70 75
80Tyr Gln Pro Ile Ser Ile Asn Tyr Arg Thr 85 904105PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
4Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1 5
10 15Pro Thr Ser Leu Leu Ile Ser Trp Ser Ala Arg Leu Lys Val Ala
Arg 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln 35 40 45Glu Phe Thr Val Pro Lys Asn Val Tyr Thr Ala Thr Ile
Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala
Val Thr Arg Phe65 70 75 80Arg Asp Tyr Gln Pro Ile Ser Ile Asn Tyr
Arg Thr Glu Ile Asp Lys 85 90 95Pro Ser Gln His His His His His His
100 105599PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 5Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr Pro Thr1 5 10 15Ser Leu Leu Ile Ser Trp Asp Ser Gly Arg
Gly Ser Tyr Gln Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly
Asn Ser Pro Val Gln Glu Phe 35 40 45Thr Val Pro Gly Pro Val His Thr
Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr
Val Tyr Ala Val Thr Asp His Lys Pro65 70 75 80His Ala Asp Gly Pro
His Thr Tyr His Glu Ser Pro Ile Ser Ile Asn 85 90 95Tyr Arg
Thr6114PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 6Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu
Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Asp Ser
Gly Arg Gly Ser Tyr Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr Val Pro Gly Pro Val
His Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr Thr
Ile Thr Val Tyr Ala Val Thr Asp His65 70 75 80Lys Pro His Ala Asp
Gly Pro His Thr Tyr His Glu Ser Pro Ile Ser 85 90 95Ile Asn Tyr Arg
Thr Glu Ile Asp Lys Pro Ser Gln His His His His 100 105 110His
His794PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 7Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr Pro Thr1 5 10 15Ser Leu Leu Ile Ser Trp Val Ala Gly Ala
Glu Asp Tyr Gln Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly
Asn Ser Pro Val Gln Glu Phe 35 40 45Thr Val Pro His Asp Leu Val Thr
Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr
Val Tyr Ala Val Thr Asp Met Met His65 70 75 80Val Glu Tyr Thr Glu
His Pro Ile Ser Ile Asn Tyr Arg Thr 85 908109PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
8Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1 5
10 15Pro Thr Ser Leu Leu Ile Ser Trp Val Ala Gly Ala Glu Asp Tyr
Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln 35 40 45Glu Phe Thr Val Pro His Asp Leu Val Thr Ala Thr Ile
Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala
Val Thr Asp Met65 70 75 80Met His Val Glu Tyr Thr Glu His Pro Ile
Ser Ile Asn Tyr Arg Thr 85 90 95Glu Ile Asp Lys Pro Ser Gln His His
His His His His 100 10597PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 9Glu Ile Asp Lys Pro Ser Gln1
5107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 10Glu Ile Asp Lys Pro Cys Gln1 51120PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 11Gly
Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser1 5 10
15Gly Ser Gly Ser 20127PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 12Pro Ser Thr Ser Thr Ser
Thr1 51310PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 13Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser1 5
101415PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 14Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser1 5 10 151520PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 15Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly1 5 10 15Gly Gly Gly Ser
201625PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 16Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly1 5 10 15Gly Gly Gly Ser Gly Gly Gly Gly Ser 20
251715PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 17Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Ser Gly1 5 10 15187PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 18Gly Pro Gly Pro Gly Pro
Gly1 51911PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 19Gly Pro Gly Pro Gly Pro Gly Pro Gly Pro Gly1 5
1020213PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 20Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr Pro Thr1 5 10 15Ser Leu Leu Ile Ser Trp Ser Ala Arg Leu
Lys Val Ala Arg Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly
Asn Ser Pro Val Gln Glu Phe 35 40 45Thr Val Pro Lys Asn Val Tyr Thr
Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr
Val Tyr Ala Val Thr Arg Phe Arg Asp65 70 75 80Tyr Gln Pro Ile Ser
Ile Asn Tyr Arg Thr Glu Ile Asp Lys Gly Ser 85 90 95Gly Ser Gly Ser
Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser 100 105 110Gly Ser
Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr 115 120
125Pro Thr Ser Leu Leu Ile Ser Trp Asp Ser Gly Arg Gly Ser Tyr Gln
130 135 140Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln145 150 155 160Glu Phe Thr Val Pro Gly Pro Val His Thr Ala
Thr Ile Ser Gly Leu 165 170 175Lys Pro Gly Val Asp Tyr Thr Ile Thr
Val Tyr Ala Val Thr Asp His 180 185 190Lys Pro His Ala Asp Gly Pro
His Thr Tyr His Glu Ser Pro Ile Ser 195 200 205Ile Asn Tyr Arg Thr
21021222PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 21Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu
Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Ser Ala
Arg Leu Lys Val Ala Arg 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr Val Pro Lys Asn Val
Tyr Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr Thr
Ile Thr Val Tyr Ala Val Thr Arg Phe65 70 75 80Arg Asp Tyr Gln Pro
Ile Ser Ile Asn Tyr Arg Thr Glu Ile Asp Lys 85 90 95Gly Ser Gly Ser
Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser 100 105 110Gly Ser
Gly Ser Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala 115 120
125Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Asp Ser Gly Arg Gly Ser
130 135 140Tyr Gln Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn
Ser Pro145 150 155 160Val Gln Glu Phe Thr Val Pro Gly Pro Val His
Thr Ala Thr Ile Ser 165 170 175Gly Leu Lys Pro Gly Val Asp Tyr Thr
Ile Thr Val Tyr Ala Val Thr 180 185 190Asp His Lys Pro His Ala Asp
Gly Pro His Thr Tyr His Glu Ser Pro 195 200 205Ile Ser Ile Asn Tyr
Arg Thr Glu Ile Asp Lys Pro Ser Gln 210 215 22022228PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
22Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1
5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Ser Ala Arg Leu Lys Val Ala
Arg 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln 35 40 45Glu Phe Thr Val Pro Lys Asn Val Tyr Thr Ala Thr Ile
Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala
Val Thr Arg Phe65 70 75 80Arg Asp Tyr Gln Pro Ile Ser Ile Asn Tyr
Arg Thr Glu Ile Asp Lys 85 90 95Gly Ser Gly Ser Gly Ser Gly Ser Gly
Ser Gly Ser Gly Ser Gly Ser 100 105 110Gly Ser Gly Ser Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ala 115 120 125Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Asp Ser Gly Arg Gly Ser 130 135 140Tyr Gln Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro145 150 155
160Val Gln Glu Phe Thr Val Pro Gly Pro Val His Thr Ala Thr Ile Ser
165 170 175Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala
Val Thr 180 185 190Asp His Lys Pro His Ala Asp Gly Pro His Thr Tyr
His Glu Ser Pro 195 200 205Ile Ser Ile Asn Tyr Arg Thr Glu Ile Asp
Lys Pro Ser Gln His His 210 215 220His His His
His22523220PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 23Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr Pro Thr1 5 10 15Ser Leu Leu Ile Ser Trp Asp Ser Gly Arg
Gly Ser Tyr Gln Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly
Asn Ser Pro Val Gln Glu Phe 35 40 45Thr Val Pro Gly Pro Val His Thr
Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr
Val Tyr Ala Val Thr Asp His Lys Pro65 70 75 80His Ala Asp Gly Pro
His Thr Tyr His Glu Ser Pro Ile Ser Ile Asn 85 90 95Tyr Arg Thr Glu
Ile Asp Lys Gly Ser Gly Ser Gly Ser Gly Ser Gly 100 105 110Ser Gly
Ser Gly Ser Gly Ser Gly Ser Gly Ser Val Ser Asp Val Pro 115 120
125Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser
130 135 140Trp Ser Ala Arg Leu Lys Val Ala Arg Tyr Tyr Arg Ile Thr
Tyr Gly145 150 155 160Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe
Thr Val Pro Lys Asn 165 170 175Val Tyr Thr Ala Thr Ile Ser Gly Leu
Lys Pro Gly Val Asp Tyr Thr 180 185 190Ile Thr Val Tyr Ala Val Thr
Arg Phe Arg Asp Tyr Gln Pro Ile Ser 195 200 205Ile Asn Tyr Arg Thr
Glu Ile Asp Lys Pro Ser Gln 210 215 22024222PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
24Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1
5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Asp Ser Gly Arg Gly Ser Tyr
Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln 35 40 45Glu Phe Thr Val Pro Gly Pro Val His Thr Ala Thr Ile
Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala
Val Thr Asp His65 70 75 80Lys Pro His Ala Asp Gly Pro His Thr Tyr
His Glu Ser Pro Ile Ser 85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys
Gly Ser Gly Ser Gly Ser Gly 100 105 110Ser Gly Ser Gly Ser Gly Ser
Gly Ser Gly Ser Gly Ser Val Ser Asp 115 120 125Val Pro Arg Asp Leu
Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu 130 135 140Ile Ser Trp
Ser Ala Arg Leu Lys Val Ala Arg Tyr Tyr Arg Ile Thr145 150 155
160Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro
165 170 175Lys Asn Val Tyr Thr Ala Thr Ile Ser Gly Leu Lys Pro Gly
Val Asp 180 185 190Tyr Thr Ile Thr Val Tyr Ala Val Thr Arg Phe Arg
Asp Tyr Gln Pro 195 200 205Ile Ser Ile Asn Tyr Arg Thr Glu Ile Asp
Lys Pro Ser Gln 210 215 22025228PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 25Met Gly Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu
Leu Ile Ser Trp Asp Ser Gly Arg Gly Ser Tyr Gln 20 25 30Tyr Tyr Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe
Thr Val Pro Gly Pro Val His Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys
Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Asp His65 70 75
80Lys Pro His Ala Asp Gly Pro His Thr Tyr His Glu Ser Pro Ile Ser
85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys Gly Ser Gly Ser Gly Ser
Gly 100 105 110Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser
Val Ser Asp 115 120 125Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr
Pro Thr Ser
Leu Leu 130 135 140Ile Ser Trp Ser Ala Arg Leu Lys Val Ala Arg Tyr
Tyr Arg Ile Thr145 150 155 160Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln Glu Phe Thr Val Pro 165 170 175Lys Asn Val Tyr Thr Ala Thr
Ile Ser Gly Leu Lys Pro Gly Val Asp 180 185 190Tyr Thr Ile Thr Val
Tyr Ala Val Thr Arg Phe Arg Asp Tyr Gln Pro 195 200 205Ile Ser Ile
Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln His His 210 215 220His
His His His22526208PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 26Val Ser Asp Val Pro Arg Asp Leu
Glu Val Val Ala Ala Thr Pro Thr1 5 10 15Ser Leu Leu Ile Ser Trp Ser
Ala Arg Leu Lys Val Ala Arg Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40 45Thr Val Pro Lys Asn
Val Tyr Thr Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr
Thr Ile Thr Val Tyr Ala Val Thr Arg Phe Arg Asp65 70 75 80Tyr Gln
Pro Ile Ser Ile Asn Tyr Arg Thr Glu Ile Asp Lys Gly Ser 85 90 95Gly
Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser 100 105
110Gly Ser Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr
115 120 125Pro Thr Ser Leu Leu Ile Ser Trp Val Ala Gly Ala Glu Asp
Tyr Gln 130 135 140Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn
Ser Pro Val Gln145 150 155 160Glu Phe Thr Val Pro His Asp Leu Val
Thr Ala Thr Ile Ser Gly Leu 165 170 175Lys Pro Gly Val Asp Tyr Thr
Ile Thr Val Tyr Ala Val Thr Asp Met 180 185 190Met His Val Glu Tyr
Thr Glu His Pro Ile Ser Ile Asn Tyr Arg Thr 195 200
20527217PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 27Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu
Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Ser Ala
Arg Leu Lys Val Ala Arg 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr Val Pro Lys Asn Val
Tyr Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr Thr
Ile Thr Val Tyr Ala Val Thr Arg Phe65 70 75 80Arg Asp Tyr Gln Pro
Ile Ser Ile Asn Tyr Arg Thr Glu Ile Asp Lys 85 90 95Gly Ser Gly Ser
Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser 100 105 110Gly Ser
Gly Ser Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala 115 120
125Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Val Ala Gly Ala Glu Asp
130 135 140Tyr Gln Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn
Ser Pro145 150 155 160Val Gln Glu Phe Thr Val Pro His Asp Leu Val
Thr Ala Thr Ile Ser 165 170 175Gly Leu Lys Pro Gly Val Asp Tyr Thr
Ile Thr Val Tyr Ala Val Thr 180 185 190Asp Met Met His Val Glu Tyr
Thr Glu His Pro Ile Ser Ile Asn Tyr 195 200 205Arg Thr Glu Ile Asp
Lys Pro Ser Gln 210 21528223PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 28Met Gly Val Ser Asp Val
Pro Arg Asp Leu Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu Leu
Ile Ser Trp Ser Ala Arg Leu Lys Val Ala Arg 20 25 30Tyr Tyr Arg Ile
Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr
Val Pro Lys Asn Val Tyr Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro
Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Arg Phe65 70 75
80Arg Asp Tyr Gln Pro Ile Ser Ile Asn Tyr Arg Thr Glu Ile Asp Lys
85 90 95Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly
Ser 100 105 110Gly Ser Gly Ser Val Ser Asp Val Pro Arg Asp Leu Glu
Val Val Ala 115 120 125Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Val
Ala Gly Ala Glu Asp 130 135 140Tyr Gln Tyr Tyr Arg Ile Thr Tyr Gly
Glu Thr Gly Gly Asn Ser Pro145 150 155 160Val Gln Glu Phe Thr Val
Pro His Asp Leu Val Thr Ala Thr Ile Ser 165 170 175Gly Leu Lys Pro
Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr 180 185 190Asp Met
Met His Val Glu Tyr Thr Glu His Pro Ile Ser Ile Asn Tyr 195 200
205Arg Thr Glu Ile Asp Lys Pro Ser Gln His His His His His His 210
215 22029208PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 29Val Ser Asp Val Pro Arg Asp Leu
Glu Val Val Ala Ala Thr Pro Thr1 5 10 15Ser Leu Leu Ile Ser Trp Val
Ala Gly Ala Glu Asp Tyr Gln Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40 45Thr Val Pro His Asp
Leu Val Thr Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr
Thr Ile Thr Val Tyr Ala Val Thr Asp Met Met His65 70 75 80Val Glu
Tyr Thr Glu His Pro Ile Ser Ile Asn Tyr Arg Thr Glu Ile 85 90 95Asp
Lys Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser 100 105
110Gly Ser Gly Ser Gly Ser Val Ser Asp Val Pro Arg Asp Leu Glu Val
115 120 125Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Ser Ala
Arg Leu 130 135 140Lys Val Ala Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn145 150 155 160Ser Pro Val Gln Glu Phe Thr Val Pro
Lys Asn Val Tyr Thr Ala Thr 165 170 175Ile Ser Gly Leu Lys Pro Gly
Val Asp Tyr Thr Ile Thr Val Tyr Ala 180 185 190Val Thr Arg Phe Arg
Asp Tyr Gln Pro Ile Ser Ile Asn Tyr Arg Thr 195 200
20530217PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 30Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu
Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Val Ala
Gly Ala Glu Asp Tyr Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr Val Pro His Asp Leu
Val Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr Thr
Ile Thr Val Tyr Ala Val Thr Asp Met65 70 75 80Met His Val Glu Tyr
Thr Glu His Pro Ile Ser Ile Asn Tyr Arg Thr 85 90 95Glu Ile Asp Lys
Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser 100 105 110Gly Ser
Gly Ser Gly Ser Gly Ser Val Ser Asp Val Pro Arg Asp Leu 115 120
125Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Ser Ala
130 135 140Arg Leu Lys Val Ala Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly145 150 155 160Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro
Lys Asn Val Tyr Thr 165 170 175Ala Thr Ile Ser Gly Leu Lys Pro Gly
Val Asp Tyr Thr Ile Thr Val 180 185 190Tyr Ala Val Thr Arg Phe Arg
Asp Tyr Gln Pro Ile Ser Ile Asn Tyr 195 200 205Arg Thr Glu Ile Asp
Lys Pro Ser Gln 210 21531223PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 31Met Gly Val Ser Asp Val
Pro Arg Asp Leu Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu Leu
Ile Ser Trp Val Ala Gly Ala Glu Asp Tyr Gln 20 25 30Tyr Tyr Arg Ile
Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr
Val Pro His Asp Leu Val Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro
Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Asp Met65 70 75
80Met His Val Glu Tyr Thr Glu His Pro Ile Ser Ile Asn Tyr Arg Thr
85 90 95Glu Ile Asp Lys Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly
Ser 100 105 110Gly Ser Gly Ser Gly Ser Gly Ser Val Ser Asp Val Pro
Arg Asp Leu 115 120 125Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu
Ile Ser Trp Ser Ala 130 135 140Arg Leu Lys Val Ala Arg Tyr Tyr Arg
Ile Thr Tyr Gly Glu Thr Gly145 150 155 160Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Lys Asn Val Tyr Thr 165 170 175Ala Thr Ile Ser
Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Val 180 185 190Tyr Ala
Val Thr Arg Phe Arg Asp Tyr Gln Pro Ile Ser Ile Asn Tyr 195 200
205Arg Thr Glu Ile Asp Lys Pro Ser Gln His His His His His His 210
215 22032146PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 32Val Ser Asp Val Pro Arg Asp Leu
Glu Val Val Ala Ala Thr Pro Thr1 5 10 15Ser Leu Leu Ile Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Tyr Tyr Arg Ile Thr Tyr Gly Glu 35 40 45Thr Gly Gly Asn Ser
Pro Val Gln Glu Phe Thr Val Xaa Xaa Xaa Xaa 50 55 60Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa65 70 75 80Ala Thr
Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Val 85 90 95Tyr
Ala Val Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 100 105
110Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
115 120 125Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ile Ser Ile
Asn Tyr 130 135 140Arg Thr1453310PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 33 Ser Trp Val Ala Gly Ala
Glu Asp Tyr Gln1 5 103418PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 34Xaa Xaa Xaa Xaa Xaa Val Ala
Gly Ala Glu Asp Tyr Gln Xaa Xaa Xaa1 5 10 15Xaa Xaa356PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 35Pro
His Asp Leu Val Thr1 53614PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 36Xaa Xaa Xaa Xaa Xaa His Asp
Leu Val Xaa Xaa Xaa Xaa Xaa1 5 103712PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 37Thr
Asp Met Met His Val Glu Tyr Thr Glu His Pro1 5 103820PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 38Xaa
Xaa Xaa Xaa Xaa Asp Met Met His Val Glu Tyr Thr Glu His Xaa1 5 10
15Xaa Xaa Xaa Xaa 203910PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 39Ser Trp Asp Ser Gly Arg Gly
Ser Tyr Gln1 5 104018PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 40Xaa Xaa Xaa Xaa Xaa Asp Ser
Gly Arg Gly Ser Tyr Gln Xaa Xaa Xaa1 5 10 15Xaa Xaa416PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 41Pro
Gly Pro Val His Thr1 54214PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 42Xaa Xaa Xaa Xaa Xaa Gly Pro
Val His Xaa Xaa Xaa Xaa Xaa1 5 104317PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 43Thr
Asp His Lys Pro His Ala Asp Gly Pro His Thr Tyr His Glu Ser1 5 10
15Pro4424PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 44Xaa Xaa Xaa Xaa Xaa Asp His Lys Pro His Ala Asp
Gly Pro His Thr1 5 10 15Tyr His Glu Xaa Xaa Xaa Xaa Xaa
204510PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 45Ser Trp Ser Ala Arg Leu Lys Val Ala Arg1 5
104617PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 46Xaa Xaa Xaa Xaa Xaa Ser Ala Arg Leu Lys Val Ala
Xaa Xaa Xaa Xaa1 5 10 15Xaa476PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 47Pro Lys Asn Val Tyr Thr1
54814PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 48Xaa Xaa Xaa Xaa Xaa Lys Asn Val Tyr Xaa Xaa Xaa
Xaa Xaa1 5 10498PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 49Thr Arg Phe Arg Asp Tyr Gln Pro1
55016PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 50Xaa Xaa Xaa Xaa Xaa Arg Phe Arg Asp Tyr Gln Xaa
Xaa Xaa Xaa Xaa1 5 10 15513PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 51Gly Pro
Gly152109PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 52Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu
Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Leu Pro
Gly Lys Leu Arg Tyr Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr Val Pro His Asp Leu
Arg Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr Thr
Ile Thr Val Tyr Ala Val Thr Asn Met65 70 75 80Met His Val Glu Tyr
Ser Glu Tyr Pro Ile Ser Ile Asn Tyr Arg Thr 85 90 95Glu Ile Asp Lys
Pro Ser Gln His His His His His His 100 10553223PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
53Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1
5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Leu Pro Gly Lys Leu Arg Tyr
Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln 35 40 45Glu Phe Thr Val Pro His Asp Leu Arg Thr Ala Thr Ile
Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala
Val Thr Asn Met65 70 75 80Met His Val Glu Tyr Ser Glu Tyr Pro Ile
Ser Ile Asn Tyr Arg Thr 85 90 95Glu Ile Asp Lys Gly Ser Gly Ser Gly
Ser Gly Ser Gly Ser Gly Ser 100 105 110Gly Ser Gly Ser Gly Ser Gly
Ser Val Ser Asp Val Pro Arg Asp Leu 115 120 125Glu Val Val Ala Ala
Thr Pro Thr Ser Leu Leu Ile Ser Trp Ser Ala 130 135 140Arg Leu Lys
Val Ala Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly145 150 155
160Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Lys Asn Val Tyr Thr
165 170 175Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile
Thr Val 180 185 190Tyr Ala Val Thr Arg Phe Arg Asp Tyr Gln Pro Ile
Ser Ile Asn Tyr 195 200 205Arg Thr Glu Ile Asp Lys Pro Cys Gln His
His His His His His 210 215 22054223PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
54Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1
5 10
15Pro Thr Ser Leu Leu Ile Ser Trp Ser Ala Arg Leu Lys Val Ala Arg
20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val
Gln 35 40 45Glu Phe Thr Val Pro Lys Asn Val Tyr Thr Ala Thr Ile Ser
Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val
Thr Arg Phe65 70 75 80Arg Asp Tyr Gln Pro Ile Ser Ile Asn Tyr Arg
Thr Glu Ile Asp Lys 85 90 95Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser
Gly Ser Gly Ser Gly Ser 100 105 110Gly Ser Gly Ser Val Ser Asp Val
Pro Arg Asp Leu Glu Val Val Ala 115 120 125Ala Thr Pro Thr Ser Leu
Leu Ile Ser Trp Leu Pro Gly Lys Leu Arg 130 135 140Tyr Gln Tyr Tyr
Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro145 150 155 160Val
Gln Glu Phe Thr Val Pro His Asp Leu Arg Thr Ala Thr Ile Ser 165 170
175Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr
180 185 190Asn Met Met His Val Glu Tyr Ser Glu Tyr Pro Ile Ser Ile
Asn Tyr 195 200 205Arg Thr Glu Ile Asp Lys Pro Cys Gln His His His
His His His 210 215 22055223PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 55Met Gly Val Ser Asp Val
Pro Arg Asp Leu Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu Leu
Ile Ser Trp Val Ala Gly Ala Glu Asp Tyr Gln 20 25 30Tyr Tyr Arg Ile
Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr
Val Pro His Asp Leu Val Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro
Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Asp Met65 70 75
80Met His Val Glu Tyr Thr Glu His Pro Ile Ser Ile Asn Tyr Arg Thr
85 90 95Glu Ile Asp Lys Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly
Ser 100 105 110Gly Ser Gly Ser Gly Ser Gly Ser Val Ser Asp Val Pro
Arg Asp Leu 115 120 125Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu
Ile Ser Trp Ser Ala 130 135 140Arg Leu Lys Val Ala Arg Tyr Tyr Arg
Ile Thr Tyr Gly Glu Thr Gly145 150 155 160Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Lys Asn Val Tyr Thr 165 170 175Ala Thr Ile Ser
Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Val 180 185 190Tyr Ala
Val Thr Arg Phe Arg Asp Tyr Gln Pro Ile Ser Ile Asn Tyr 195 200
205Arg Thr Glu Ile Asp Lys Pro Cys Gln His His His His His His 210
215 22056228PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 56Met Gly Val Ser Asp Val Pro Arg
Asp Leu Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu Leu Ile Ser
Trp Asp Ser Gly Arg Gly Ser Tyr Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr
Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr Val Pro
Gly Pro Val His Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro Gly Val
Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Asp His65 70 75 80Lys Pro
His Ala Asp Gly Pro His Thr Tyr His Glu Ser Pro Ile Ser 85 90 95Ile
Asn Tyr Arg Thr Glu Ile Asp Lys Gly Ser Gly Ser Gly Ser Gly 100 105
110Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Val Ser Asp
115 120 125Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu 130 135 140Ile Ser Trp Ser Ala Arg Leu Lys Val Ala Arg Tyr
Tyr Arg Ile Thr145 150 155 160Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln Glu Phe Thr Val Pro 165 170 175Lys Asn Val Tyr Thr Ala Thr
Ile Ser Gly Leu Lys Pro Gly Val Asp 180 185 190Tyr Thr Ile Thr Val
Tyr Ala Val Thr Arg Phe Arg Asp Tyr Gln Pro 195 200 205Ile Ser Ile
Asn Tyr Arg Thr Glu Ile Asp Lys Pro Cys Gln His His 210 215 220His
His His His22557223PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 57Met Gly Val Ser Asp Val Pro Arg
Asp Leu Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu Leu Ile Ser
Trp Ser Ala Arg Leu Lys Val Ala Arg 20 25 30Tyr Tyr Arg Ile Thr Tyr
Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr Val Pro
Lys Asn Val Tyr Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro Gly Val
Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Arg Phe65 70 75 80Arg Asp
Tyr Gln Pro Ile Ser Ile Asn Tyr Arg Thr Glu Ile Asp Lys 85 90 95Gly
Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser 100 105
110Gly Ser Gly Ser Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala
115 120 125Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Val Ala Gly Ala
Glu Asp 130 135 140Tyr Gln Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly
Gly Asn Ser Pro145 150 155 160Val Gln Glu Phe Thr Val Pro His Asp
Leu Val Thr Ala Thr Ile Ser 165 170 175Gly Leu Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Val Thr 180 185 190Asp Met Met His Val
Glu Tyr Thr Glu His Pro Ile Ser Ile Asn Tyr 195 200 205Arg Thr Glu
Ile Asp Lys Pro Cys Gln His His His His His His 210 215
22058228PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 58Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu
Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Ser Ala
Arg Leu Lys Val Ala Arg 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr Val Pro Lys Asn Val
Tyr Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr Thr
Ile Thr Val Tyr Ala Val Thr Arg Phe65 70 75 80Arg Asp Tyr Gln Pro
Ile Ser Ile Asn Tyr Arg Thr Glu Ile Asp Lys 85 90 95Gly Ser Gly Ser
Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser 100 105 110Gly Ser
Gly Ser Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala 115 120
125Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Asp Ser Gly Arg Gly Ser
130 135 140Tyr Gln Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn
Ser Pro145 150 155 160Val Gln Glu Phe Thr Val Pro Gly Pro Val His
Thr Ala Thr Ile Ser 165 170 175Gly Leu Lys Pro Gly Val Asp Tyr Thr
Ile Thr Val Tyr Ala Val Thr 180 185 190Asp His Lys Pro His Ala Asp
Gly Pro His Thr Tyr His Glu Ser Pro 195 200 205Ile Ser Ile Asn Tyr
Arg Thr Glu Ile Asp Lys Pro Cys Gln His His 210 215 220His His His
His2255910PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 59Ser Trp Leu Pro Gly Lys Leu Arg Tyr Gln1 5
106018PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 60Xaa Xaa Xaa Xaa Xaa Leu Pro Gly Lys Leu Arg Tyr
Gln Xaa Xaa Xaa1 5 10 15Xaa Xaa616PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 61Pro His Asp Leu Arg Thr1
56214PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 62Xaa Xaa Xaa Xaa Xaa His Asp Leu Arg Xaa Xaa Xaa
Xaa Xaa1 5 106312PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 63Thr Asn Met Met His Val Glu Tyr Ser
Glu Tyr Pro1 5 106420PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 64Xaa Xaa Xaa Xaa Xaa Asn Met
Met His Val Glu Tyr Ser Glu Tyr Xaa1 5 10 15Xaa Xaa Xaa Xaa
206582PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 65Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu
Ile Ser Trp Ser Ala1 5 10 15Arg Leu Lys Val Ala Arg Tyr Tyr Arg Ile
Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr
Val Pro Lys Asn Val Tyr Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro
Gly Val Asp Tyr Thr Ile Thr Val 50 55 60Tyr Ala Val Thr Arg Phe Arg
Asp Tyr Gln Pro Ile Ser Ile Asn Tyr65 70 75 80Arg
Thr6686PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 66Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu
Ile Ser Trp Val Ala1 5 10 15Gly Ala Glu Asp Tyr Gln Tyr Tyr Arg Ile
Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr
Val Pro His Asp Leu Val Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro
Gly Val Asp Tyr Thr Ile Thr Val 50 55 60Tyr Ala Val Thr Asp Met Met
His Val Glu Tyr Thr Glu His Pro Ile65 70 75 80Ser Ile Asn Tyr Arg
Thr 856791PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 67Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu
Ile Ser Trp Asp Ser1 5 10 15Gly Arg Gly Ser Tyr Gln Tyr Tyr Arg Ile
Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr
Val Pro Gly Pro Val His Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro
Gly Val Asp Tyr Thr Ile Thr Val 50 55 60Tyr Ala Val Thr Asp His Lys
Pro His Ala Asp Gly Pro His Thr Tyr65 70 75 80His Glu Ser Pro Ile
Ser Ile Asn Tyr Arg Thr 85 906886PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 68Glu Val Val Ala Ala
Thr Pro Thr Ser Leu Leu Ile Ser Trp Leu Pro1 5 10 15Gly Lys Leu Arg
Tyr Gln Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser
Pro Val Gln Glu Phe Thr Val Pro His Asp Leu Arg Thr 35 40 45Ala Thr
Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Val 50 55 60Tyr
Ala Val Thr Asn Met Met His Val Glu Tyr Ser Glu Tyr Pro Ile65 70 75
80Ser Ile Asn Tyr Arg Thr 856910PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 69Met Gly Val Ser Asp Val
Pro Arg Asp Leu1 5 10709PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 70Gly Val Ser Asp Val Pro Arg
Asp Leu1 5718PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 71Val Ser Asp Val Pro Arg Asp Leu1
5729PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 72Xaa Xaa Ser Asp Val Pro Arg Asp Leu1
5738PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 73Xaa Xaa Asp Val Pro Arg Asp Leu1
5747PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 74Xaa Xaa Val Pro Arg Asp Leu1 5756PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 75Xaa
Xaa Pro Arg Asp Leu1 5765PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 76Xaa Xaa Arg Asp Leu1
5774PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 77Xaa Xaa Asp Leu1784PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 78Glu
Ile Asp Lys1795PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 79Glu Ile Asp Lys Pro1 5806PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 80Glu
Ile Asp Lys Pro Ser1 5816PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 81Glu Ile Asp Lys Pro Cys1
58294PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 82Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr Pro Thr1 5 10 15Ser Leu Leu Ile Ser Trp Leu Pro Gly Lys
Leu Arg Tyr Gln Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly
Asn Ser Pro Val Gln Glu Phe 35 40 45Thr Val Pro His Asp Leu Arg Thr
Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr
Val Tyr Ala Val Thr Asn Met Met His65 70 75 80Val Glu Tyr Ser Glu
Tyr Pro Ile Ser Ile Asn Tyr Arg Thr 85 9083185PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
83Glu Val Val Ala Ala Thr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1
5 10 15Xaa Xaa Xaa Xaa Xaa Ser Leu Leu Ile Xaa Xaa Xaa Xaa Xaa Xaa
Xaa 20 25 30Xaa Xaa Xaa Ser Trp Val Ala Gly Ala Glu Asp Tyr Gln Xaa
Xaa Xaa 35 40 45Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr Tyr Arg Ile Thr Tyr
Gly Glu Xaa 50 55 60Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa65 70 75 80Xaa Xaa Xaa Gln Glu Phe Thr Val Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95Xaa Xaa Pro His Asp Leu Val Thr Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 100 105 110Xaa Xaa Ala Thr Ile Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 115 120 125Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Asp Tyr Thr Ile Thr Val Tyr 130 135 140Ala Val Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Thr Asp Met Met145 150 155
160His Val Glu Tyr Thr Glu His Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
165 170 175Xaa Xaa Ile Ser Ile Asn Tyr Arg Thr 180
18584190PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 84Glu Val Val Ala Ala Thr Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa1 5 10 15Xaa Xaa Xaa Xaa Xaa Ser Leu Leu Ile Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30Xaa Xaa Xaa Ser Trp Asp Ser Gly Arg
Gly Ser Tyr Gln Xaa Xaa Xaa 35 40 45Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr
Tyr Arg Ile Thr Tyr Gly Glu Xaa 50 55 60Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa65 70 75 80Xaa Xaa Xaa Gln Glu
Phe Thr Val Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95Xaa Xaa Pro Gly
Pro Val His Thr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 100 105 110Xaa Xaa
Ala Thr Ile Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 115 120
125Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asp Tyr Thr Ile Thr Val Tyr
130 135 140Ala Val Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Thr Asp
His Lys145 150 155 160Pro His Ala Asp Gly Pro His Thr Tyr His Glu
Ser Pro Xaa Xaa Xaa 165 170 175Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ile Ser
Ile Asn Tyr Arg Thr 180 185 19085185PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
85Glu Val Val Ala Ala Thr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1
5 10 15Xaa Xaa Xaa Xaa Xaa Ser Leu Leu Ile Xaa Xaa Xaa Xaa Xaa Xaa
Xaa 20 25
30Xaa Xaa Xaa Ser Trp Leu Pro Gly Lys Leu Arg Tyr Gln Xaa Xaa Xaa
35 40 45Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr Tyr Arg Ile Thr Tyr Gly Glu
Xaa 50 55 60Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa65 70 75 80Xaa Xaa Xaa Gln Glu Phe Thr Val Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 85 90 95Xaa Xaa Pro His Asp Leu Arg Thr Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa 100 105 110Xaa Xaa Ala Thr Ile Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 115 120 125Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Asp Tyr Thr Ile Thr Val Tyr 130 135 140Ala Val Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Thr Asn Met Met145 150 155 160His Val
Glu Tyr Ser Glu Tyr Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 165 170
175Xaa Xaa Ile Ser Ile Asn Tyr Arg Thr 180 18586181PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
86Glu Val Val Ala Ala Thr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1
5 10 15Xaa Xaa Xaa Xaa Xaa Ser Leu Leu Ile Xaa Xaa Xaa Xaa Xaa Xaa
Xaa 20 25 30Xaa Xaa Xaa Ser Trp Ser Ala Arg Leu Lys Val Ala Arg Xaa
Xaa Xaa 35 40 45Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr Tyr Arg Ile Thr Tyr
Gly Glu Xaa 50 55 60Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa65 70 75 80Xaa Xaa Xaa Gln Glu Phe Thr Val Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95Xaa Xaa Pro Lys Asn Val Tyr Thr Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 100 105 110Xaa Xaa Ala Thr Ile Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 115 120 125Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Asp Tyr Thr Ile Thr Val Tyr 130 135 140Ala Val Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Thr Arg Phe Arg145 150 155
160Asp Tyr Gln Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ile Ser
165 170 175Ile Asn Tyr Arg Thr 18087215PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
87Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr1
5 10 15Ser Leu Leu Ile Ser Trp Leu Pro Gly Lys Leu Arg Tyr Gln Tyr
Tyr 20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln
Glu Phe 35 40 45Thr Val Pro His Asp Leu Arg Thr Ala Thr Ile Ser Gly
Leu Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr
Asn Met Met His65 70 75 80Val Glu Tyr Ser Glu Tyr Pro Ile Ser Ile
Asn Tyr Arg Thr Glu Ile 85 90 95Asp Lys Gly Ser Gly Ser Gly Ser Gly
Ser Gly Ser Gly Ser Gly Ser 100 105 110Gly Ser Gly Ser Gly Ser Val
Ser Asp Val Pro Arg Asp Leu Glu Val 115 120 125Val Ala Ala Thr Pro
Thr Ser Leu Leu Ile Ser Trp Ser Ala Arg Leu 130 135 140Lys Val Ala
Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn145 150 155
160Ser Pro Val Gln Glu Phe Thr Val Pro Lys Asn Val Tyr Thr Ala Thr
165 170 175Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Val
Tyr Ala 180 185 190Val Thr Arg Phe Arg Asp Tyr Gln Pro Ile Ser Ile
Asn Tyr Arg Thr 195 200 205Glu Ile Asp Lys Pro Cys Gln 210
21588215PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 88Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr Pro Thr1 5 10 15Ser Leu Leu Ile Ser Trp Ser Ala Arg Leu
Lys Val Ala Arg Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly
Asn Ser Pro Val Gln Glu Phe 35 40 45Thr Val Pro Lys Asn Val Tyr Thr
Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr
Val Tyr Ala Val Thr Arg Phe Arg Asp65 70 75 80Tyr Gln Pro Ile Ser
Ile Asn Tyr Arg Thr Glu Ile Asp Lys Gly Ser 85 90 95Gly Ser Gly Ser
Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser 100 105 110Gly Ser
Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr 115 120
125Pro Thr Ser Leu Leu Ile Ser Trp Leu Pro Gly Lys Leu Arg Tyr Gln
130 135 140Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln145 150 155 160Glu Phe Thr Val Pro His Asp Leu Arg Thr Ala
Thr Ile Ser Gly Leu 165 170 175Lys Pro Gly Val Asp Tyr Thr Ile Thr
Val Tyr Ala Val Thr Asn Met 180 185 190Met His Val Glu Tyr Ser Glu
Tyr Pro Ile Ser Ile Asn Tyr Arg Thr 195 200 205Glu Ile Asp Lys Pro
Cys Gln 210 21589215PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 89Val Ser Asp Val Pro Arg Asp Leu
Glu Val Val Ala Ala Thr Pro Thr1 5 10 15Ser Leu Leu Ile Ser Trp Val
Ala Gly Ala Glu Asp Tyr Gln Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40 45Thr Val Pro His Asp
Leu Val Thr Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr
Thr Ile Thr Val Tyr Ala Val Thr Asp Met Met His65 70 75 80Val Glu
Tyr Thr Glu His Pro Ile Ser Ile Asn Tyr Arg Thr Glu Ile 85 90 95Asp
Lys Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser 100 105
110Gly Ser Gly Ser Gly Ser Val Ser Asp Val Pro Arg Asp Leu Glu Val
115 120 125Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Ser Ala
Arg Leu 130 135 140Lys Val Ala Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn145 150 155 160Ser Pro Val Gln Glu Phe Thr Val Pro
Lys Asn Val Tyr Thr Ala Thr 165 170 175Ile Ser Gly Leu Lys Pro Gly
Val Asp Tyr Thr Ile Thr Val Tyr Ala 180 185 190Val Thr Arg Phe Arg
Asp Tyr Gln Pro Ile Ser Ile Asn Tyr Arg Thr 195 200 205Glu Ile Asp
Lys Pro Cys Gln 210 21590220PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 90Val Ser Asp Val Pro Arg
Asp Leu Glu Val Val Ala Ala Thr Pro Thr1 5 10 15Ser Leu Leu Ile Ser
Trp Asp Ser Gly Arg Gly Ser Tyr Gln Tyr Tyr 20 25 30Arg Ile Thr Tyr
Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40 45Thr Val Pro
Gly Pro Val His Thr Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly Val
Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Asp His Lys Pro65 70 75
80His Ala Asp Gly Pro His Thr Tyr His Glu Ser Pro Ile Ser Ile Asn
85 90 95Tyr Arg Thr Glu Ile Asp Lys Gly Ser Gly Ser Gly Ser Gly Ser
Gly 100 105 110Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Val Ser
Asp Val Pro 115 120 125Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr
Ser Leu Leu Ile Ser 130 135 140Trp Ser Ala Arg Leu Lys Val Ala Arg
Tyr Tyr Arg Ile Thr Tyr Gly145 150 155 160Glu Thr Gly Gly Asn Ser
Pro Val Gln Glu Phe Thr Val Pro Lys Asn 165 170 175Val Tyr Thr Ala
Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr 180 185 190Ile Thr
Val Tyr Ala Val Thr Arg Phe Arg Asp Tyr Gln Pro Ile Ser 195 200
205Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Cys Gln 210 215
22091215PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 91Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr Pro Thr1 5 10 15Ser Leu Leu Ile Ser Trp Ser Ala Arg Leu
Lys Val Ala Arg Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly
Asn Ser Pro Val Gln Glu Phe 35 40 45Thr Val Pro Lys Asn Val Tyr Thr
Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr
Val Tyr Ala Val Thr Arg Phe Arg Asp65 70 75 80Tyr Gln Pro Ile Ser
Ile Asn Tyr Arg Thr Glu Ile Asp Lys Gly Ser 85 90 95Gly Ser Gly Ser
Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser 100 105 110Gly Ser
Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr 115 120
125Pro Thr Ser Leu Leu Ile Ser Trp Val Ala Gly Ala Glu Asp Tyr Gln
130 135 140Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln145 150 155 160Glu Phe Thr Val Pro His Asp Leu Val Thr Ala
Thr Ile Ser Gly Leu 165 170 175Lys Pro Gly Val Asp Tyr Thr Ile Thr
Val Tyr Ala Val Thr Asp Met 180 185 190Met His Val Glu Tyr Thr Glu
His Pro Ile Ser Ile Asn Tyr Arg Thr 195 200 205Glu Ile Asp Lys Pro
Cys Gln 210 21592220PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 92Val Ser Asp Val Pro Arg Asp Leu
Glu Val Val Ala Ala Thr Pro Thr1 5 10 15Ser Leu Leu Ile Ser Trp Ser
Ala Arg Leu Lys Val Ala Arg Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40 45Thr Val Pro Lys Asn
Val Tyr Thr Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr
Thr Ile Thr Val Tyr Ala Val Thr Arg Phe Arg Asp65 70 75 80Tyr Gln
Pro Ile Ser Ile Asn Tyr Arg Thr Glu Ile Asp Lys Gly Ser 85 90 95Gly
Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser 100 105
110Gly Ser Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr
115 120 125Pro Thr Ser Leu Leu Ile Ser Trp Asp Ser Gly Arg Gly Ser
Tyr Gln 130 135 140Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn
Ser Pro Val Gln145 150 155 160Glu Phe Thr Val Pro Gly Pro Val His
Thr Ala Thr Ile Ser Gly Leu 165 170 175Lys Pro Gly Val Asp Tyr Thr
Ile Thr Val Tyr Ala Val Thr Asp His 180 185 190Lys Pro His Ala Asp
Gly Pro His Thr Tyr His Glu Ser Pro Ile Ser 195 200 205Ile Asn Tyr
Arg Thr Glu Ile Asp Lys Pro Cys Gln 210 215 220936PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 93Pro
Ala Pro Ala Pro Ala1 59412PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 94Pro Ala Pro Ala Pro Ala Pro
Ala Pro Ala Pro Ala1 5 109518PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 95Pro Ala Pro Ala Pro Ala Pro
Ala Pro Ala Pro Ala Pro Ala Pro Ala1 5 10 15Pro Ala965PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 96Glu
Gly Ser Gly Ser1 5975PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 97Glu Gly Ser Gly Cys1
598208PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 98Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr Pro Thr1 5 10 15Ser Leu Leu Ile Ser Trp Leu Pro Gly Lys
Leu Arg Tyr Gln Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly
Asn Ser Pro Val Gln Glu Phe 35 40 45Thr Val Pro His Asp Leu Arg Thr
Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr
Val Tyr Ala Val Thr Asn Met Met His65 70 75 80Val Glu Tyr Ser Glu
Tyr Pro Ile Ser Ile Asn Tyr Arg Thr Glu Pro 85 90 95Ala Pro Ala Pro
Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro 100 105 110Ala Val
Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro 115 120
125Thr Ser Leu Leu Ile Ser Trp Ser Ala Arg Leu Lys Val Ala Arg Tyr
130 135 140Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val
Gln Glu145 150 155 160Phe Thr Val Pro Lys Asn Val Tyr Thr Ala Thr
Ile Ser Gly Leu Lys 165 170 175Pro Gly Val Asp Tyr Thr Ile Thr Val
Tyr Ala Val Thr Arg Phe Arg 180 185 190Asp Tyr Gln Pro Ile Ser Ile
Asn Tyr Arg Thr Glu Gly Ser Gly Xaa 195 200 20599208PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
99Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr1
5 10 15Ser Leu Leu Ile Ser Trp Ser Ala Arg Leu Lys Val Ala Arg Tyr
Tyr 20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln
Glu Phe 35 40 45Thr Val Pro Lys Asn Val Tyr Thr Ala Thr Ile Ser Gly
Leu Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr
Arg Phe Arg Asp65 70 75 80Tyr Gln Pro Ile Ser Ile Asn Tyr Arg Thr
Glu Pro Ala Pro Ala Pro 85 90 95Ala Pro Ala Pro Ala Pro Ala Pro Ala
Pro Ala Pro Ala Val Ser Asp 100 105 110Val Pro Arg Asp Leu Glu Val
Val Ala Ala Thr Pro Thr Ser Leu Leu 115 120 125Ile Ser Trp Leu Pro
Gly Lys Leu Arg Tyr Gln Tyr Tyr Arg Ile Thr 130 135 140Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro145 150 155
160His Asp Leu Arg Thr Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp
165 170 175Tyr Thr Ile Thr Val Tyr Ala Val Thr Asn Met Met His Val
Glu Tyr 180 185 190Ser Glu Tyr Pro Ile Ser Ile Asn Tyr Arg Thr Glu
Gly Ser Gly Xaa 195 200 205100208PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 100Val Ser Asp Val Pro
Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr1 5 10 15Ser Leu Leu Ile
Ser Trp Val Ala Gly Ala Glu Asp Tyr Gln Tyr Tyr 20 25 30Arg Ile Thr
Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40 45Thr Val
Pro His Asp Leu Val Thr Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly
Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Asp Met Met His65 70 75
80Val Glu Tyr Thr Glu His Pro Ile Ser Ile Asn Tyr Arg Thr Glu Pro
85 90 95Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala
Pro 100 105 110Ala Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala
Ala Thr Pro 115 120 125Thr Ser Leu Leu Ile Ser Trp Ser Ala Arg Leu
Lys Val Ala Arg Tyr 130 135 140Tyr Arg Ile Thr Tyr Gly Glu Thr Gly
Gly Asn Ser Pro Val Gln Glu145 150 155 160Phe Thr Val Pro Lys Asn
Val Tyr Thr Ala Thr Ile Ser Gly Leu Lys 165 170 175Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Val Thr Arg Phe Arg 180 185
190Asp Tyr Gln Pro Ile Ser Ile Asn Tyr Arg Thr Glu Gly Ser Gly Xaa
195 200 205101213PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 101Val Ser Asp Val Pro Arg Asp Leu
Glu Val Val Ala Ala Thr Pro Thr1 5 10 15Ser Leu Leu Ile Ser Trp Asp
Ser Gly Arg Gly Ser Tyr Gln Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40 45Thr Val Pro Gly Pro
Val His Thr Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr
Thr Ile Thr Val Tyr Ala Val Thr Asp His Lys Pro65 70 75 80His Ala
Asp Gly Pro His Thr Tyr His Glu Ser Pro Ile Ser Ile Asn 85 90 95Tyr
Arg Thr Glu Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala 100 105
110Pro Ala Pro Ala Pro Ala Val Ser Asp Val Pro Arg Asp Leu Glu Val
115 120 125Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Ser Ala
Arg Leu 130 135 140Lys Val Ala Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn145 150 155 160Ser Pro Val Gln Glu Phe Thr Val Pro
Lys Asn Val Tyr Thr Ala Thr 165 170 175Ile Ser Gly Leu Lys Pro Gly
Val Asp Tyr Thr Ile Thr Val Tyr Ala 180 185 190Val Thr Arg Phe Arg
Asp Tyr Gln Pro Ile Ser Ile Asn Tyr Arg Thr 195 200 205Glu Gly Ser
Gly Xaa 210102208PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 102Val Ser Asp Val Pro Arg Asp Leu
Glu Val Val Ala Ala Thr Pro Thr1 5 10 15Ser Leu Leu Ile Ser Trp Ser
Ala Arg Leu Lys Val Ala Arg Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40 45Thr Val Pro Lys Asn
Val Tyr Thr Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr
Thr Ile Thr Val Tyr Ala Val Thr Arg Phe Arg Asp65 70 75 80Tyr Gln
Pro Ile Ser Ile Asn Tyr Arg Thr Glu Pro Ala Pro Ala Pro 85 90 95Ala
Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Val Ser Asp 100 105
110Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu
115 120 125Ile Ser Trp Val Ala Gly Ala Glu Asp Tyr Gln Tyr Tyr Arg
Ile Thr 130 135 140Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu
Phe Thr Val Pro145 150 155 160His Asp Leu Val Thr Ala Thr Ile Ser
Gly Leu Lys Pro Gly Val Asp 165 170 175Tyr Thr Ile Thr Val Tyr Ala
Val Thr Asp Met Met His Val Glu Tyr 180 185 190Thr Glu His Pro Ile
Ser Ile Asn Tyr Arg Thr Glu Gly Ser Gly Xaa 195 200
205103213PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 103Val Ser Asp Val Pro Arg Asp Leu Glu Val
Val Ala Ala Thr Pro Thr1 5 10 15Ser Leu Leu Ile Ser Trp Ser Ala Arg
Leu Lys Val Ala Arg Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly
Gly Asn Ser Pro Val Gln Glu Phe 35 40 45Thr Val Pro Lys Asn Val Tyr
Thr Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile
Thr Val Tyr Ala Val Thr Arg Phe Arg Asp65 70 75 80Tyr Gln Pro Ile
Ser Ile Asn Tyr Arg Thr Glu Pro Ala Pro Ala Pro 85 90 95Ala Pro Ala
Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Val Ser Asp 100 105 110Val
Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu 115 120
125Ile Ser Trp Asp Ser Gly Arg Gly Ser Tyr Gln Tyr Tyr Arg Ile Thr
130 135 140Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe Thr
Val Pro145 150 155 160Gly Pro Val His Thr Ala Thr Ile Ser Gly Leu
Lys Pro Gly Val Asp 165 170 175Tyr Thr Ile Thr Val Tyr Ala Val Thr
Asp His Lys Pro His Ala Asp 180 185 190Gly Pro His Thr Tyr His Glu
Ser Pro Ile Ser Ile Asn Tyr Arg Thr 195 200 205Glu Gly Ser Gly Xaa
210104215PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 104Val Ser Asp Val Pro Arg Asp Leu Glu Val
Val Ala Ala Thr Pro Thr1 5 10 15Ser Leu Leu Ile Ser Trp Leu Pro Gly
Lys Leu Arg Tyr Gln Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly
Gly Asn Ser Pro Val Gln Glu Phe 35 40 45Thr Val Pro His Asp Leu Arg
Thr Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile
Thr Val Tyr Ala Val Thr Asn Met Met His65 70 75 80Val Glu Tyr Ser
Glu Tyr Pro Ile Ser Ile Asn Tyr Arg Thr Glu Ile 85 90 95Asp Lys Gly
Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser 100 105 110Gly
Ser Gly Ser Gly Ser Val Ser Asp Val Pro Arg Asp Leu Glu Val 115 120
125Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Ser Ala Arg Leu
130 135 140Lys Val Ala Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly
Gly Asn145 150 155 160Ser Pro Val Gln Glu Phe Thr Val Pro Lys Asn
Val Tyr Thr Ala Thr 165 170 175Ile Ser Gly Leu Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala 180 185 190Val Thr Arg Phe Arg Asp Tyr
Gln Pro Ile Ser Ile Asn Tyr Arg Thr 195 200 205Glu Ile Asp Lys Pro
Ser Gln 210 215105215PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 105Val Ser Asp Val Pro
Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr1 5 10 15Ser Leu Leu Ile
Ser Trp Ser Ala Arg Leu Lys Val Ala Arg Tyr Tyr 20 25 30Arg Ile Thr
Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40 45Thr Val
Pro Lys Asn Val Tyr Thr Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly
Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Arg Phe Arg Asp65 70 75
80Tyr Gln Pro Ile Ser Ile Asn Tyr Arg Thr Glu Ile Asp Lys Gly Ser
85 90 95Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly
Ser 100 105 110Gly Ser Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 115 120 125Pro Thr Ser Leu Leu Ile Ser Trp Leu Pro Gly
Lys Leu Arg Tyr Gln 130 135 140Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln145 150 155 160Glu Phe Thr Val Pro His
Asp Leu Arg Thr Ala Thr Ile Ser Gly Leu 165 170 175Lys Pro Gly Val
Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Asn Met 180 185 190Met His
Val Glu Tyr Ser Glu Tyr Pro Ile Ser Ile Asn Tyr Arg Thr 195 200
205Glu Ile Asp Lys Pro Ser Gln 210 21510699PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
106Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr1
5 10 15Ser Leu Leu Ile Ser Trp His Glu Arg Asp Gly Ser Arg Gln Tyr
Tyr 20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln
Glu Phe 35 40 45Thr Val Pro Gly Gly Val Arg Thr Ala Thr Ile Ser Gly
Leu Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr
Asp Tyr Phe Asn65 70 75 80Pro Thr Thr His Glu Tyr Ile Tyr Gln Thr
Thr Pro Ile Ser Ile Asn 85 90 95Tyr Arg Thr 107114PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
107Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1
5 10 15Pro Thr Ser Leu Leu Ile Ser Trp His Glu Arg Asp Gly Ser Arg
Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln 35 40 45Glu Phe Thr Val Pro Gly Gly Val Arg Thr Ala Thr Ile
Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala
Val Thr Asp Tyr65 70 75 80Phe Asn Pro Thr Thr His Glu Tyr Ile Tyr
Gln Thr Thr Pro Ile Ser 85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys
Pro Ser Gln His His His His 100 105 110His His10891PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
108Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp His Glu1
5 10 15Arg Asp Gly Ser Arg Gln Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Gly Gly Val
Arg Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr
Ile Thr Val 50 55 60Tyr Ala Val Thr Asp Tyr Phe Asn Pro Thr Thr His
Glu Tyr Ile Tyr65 70 75 80Gln Thr Thr Pro Ile Ser Ile Asn Tyr Arg
Thr 85 9010910PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 109Ser Trp His Glu Arg Asp Gly Ser Arg
Gln1 5 101106PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 110Pro Gly Gly Val Arg Thr1
511117PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 111Thr Asp Tyr Phe Asn Pro Thr Thr His Glu Tyr
Ile Tyr Gln Thr Thr1 5 10 15Pro11299PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
112Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr1
5 10 15Ser Leu Leu Ile Ser Trp Trp Ala Pro Val Asp Arg Tyr Gln Tyr
Tyr 20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln
Glu Phe 35 40 45Thr Val Pro Arg Asp Val Tyr Thr Ala Thr Ile Ser Gly
Leu Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr
Asp Tyr Lys Pro65 70 75 80His Ala Asp Gly Pro His Thr Tyr His Glu
Ser Pro Ile Ser Ile Asn 85 90 95Tyr Arg Thr113108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
113Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1
5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Trp Ala Pro Val Asp Arg Tyr
Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln 35 40 45Glu Phe Thr Val Pro Arg Asp Val Tyr Thr Ala Thr Ile
Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala
Val Thr Asp Tyr65 70 75 80Lys Pro His Ala Asp Gly Pro His Thr Tyr
His Glu Ser Pro Ile Ser 85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys
Pro Xaa Gln 100 10511491PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 114Glu Val Val Ala Ala
Thr Pro Thr Ser Leu Leu Ile Ser Trp Trp Ala1 5 10 15Pro Val Asp Arg
Tyr Gln Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser
Pro Val Gln Glu Phe Thr Val Pro Arg Asp Val Tyr Thr 35 40 45Ala Thr
Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Val 50 55 60Tyr
Ala Val Thr Asp Tyr Lys Pro His Ala Asp Gly Pro His Thr Tyr65 70 75
80His Glu Ser Pro Ile Ser Ile Asn Tyr Arg Thr 85
9011510PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 115Ser Trp Trp Ala Pro Val Asp Arg Tyr Gln1 5
101166PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 116Pro Arg Asp Val Tyr Thr1 511717PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 117Thr
Asp Tyr Lys Pro His Ala Asp Gly Pro His Thr Tyr His Glu Ser1 5 10
15Pro118213PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 118Val Ser Asp Val Pro Arg Asp Leu Glu Val
Val Ala Ala Thr Pro Thr1 5 10 15Ser Leu Leu Ile Ser Trp His Glu Arg
Asp Gly Ser Arg Gln Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly
Gly Asn Ser Pro Val Gln Glu Phe 35 40 45Thr Val Pro Gly Gly Val Arg
Thr Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile
Thr Val Tyr Ala Val Thr Asp Tyr Phe Asn65 70 75 80Pro Thr Thr His
Glu Tyr Ile Tyr Gln Thr Thr Pro Ile Ser Ile Asn 85 90 95Tyr Arg Thr
Glu Ile Asp Lys Gly Ser Gly Ser Gly Ser Gly Ser Gly 100 105 110Ser
Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Val Ser Asp Val Pro 115 120
125Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser
130 135 140Trp Ser Ala Arg Leu Lys Val Ala Arg Tyr Tyr Arg Ile Thr
Tyr Gly145 150 155 160Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe
Thr Val Pro Lys Asn 165 170 175Val Tyr Thr Ala Thr Ile Ser Gly Leu
Lys Pro Gly Val Asp Tyr Thr 180 185 190Ile Thr Val Tyr Ala Val Thr
Arg Phe Arg Asp Tyr Gln Pro Ile Ser 195 200 205Ile Asn Tyr Arg Thr
210119220PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 119Val Ser Asp Val Pro Arg Asp Leu Glu Val
Val Ala Ala Thr Pro Thr1 5 10 15Ser Leu Leu Ile Ser Trp His Glu Arg
Asp Gly Ser Arg Gln Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly
Gly Asn Ser Pro Val Gln Glu Phe 35 40 45Thr Val Pro Gly Gly Val Arg
Thr Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile
Thr Val Tyr Ala Val Thr Asp Tyr Phe Asn65 70 75 80Pro Thr Thr His
Glu Tyr Ile Tyr Gln Thr Thr Pro Ile Ser Ile Asn 85 90 95Tyr Arg Thr
Glu Ile Asp Lys Gly Ser Gly Ser Gly Ser Gly Ser Gly 100 105 110Ser
Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Val Ser Asp Val Pro 115 120
125Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser
130 135 140Trp Ser Ala Arg Leu Lys Val Ala Arg Tyr Tyr Arg Ile Thr
Tyr Gly145 150 155 160Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe
Thr Val Pro Lys Asn 165 170 175Val Tyr Thr Ala Thr Ile Ser Gly Leu
Lys Pro Gly Val Asp Tyr Thr 180 185 190Ile Thr Val Tyr Ala Val Thr
Arg Phe Arg Asp Tyr Gln Pro Ile Ser 195 200 205Ile Asn Tyr Arg Thr
Glu Ile Asp Lys Pro Xaa Gln 210 215 220120228PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
120Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1
5 10 15Pro Thr Ser Leu Leu Ile Ser Trp His Glu Arg Asp Gly Ser Arg
Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln 35 40 45Glu Phe Thr Val Pro Gly Gly Val Arg Thr Ala
Thr Ile Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr Thr Ile Thr Val
Tyr Ala Val Thr Asp Tyr65 70 75 80Phe Asn Pro Thr Thr His Glu Tyr
Ile Tyr Gln Thr Thr Pro Ile Ser 85 90 95Ile Asn Tyr Arg Thr Glu Ile
Asp Lys Gly Ser Gly Ser Gly Ser Gly 100 105 110Ser Gly Ser Gly Ser
Gly Ser Gly Ser Gly Ser Gly Ser Val Ser Asp 115 120 125Val Pro Arg
Asp Leu Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu 130 135 140Ile
Ser Trp Ser Ala Arg Leu Lys Val Ala Arg Tyr Tyr Arg Ile Thr145 150
155 160Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe Thr Val
Pro 165 170 175Lys Asn Val Tyr Thr Ala Thr Ile Ser Gly Leu Lys Pro
Gly Val Asp 180 185 190Tyr Thr Ile Thr Val Tyr Ala Val Thr Arg Phe
Arg Asp Tyr Gln Pro 195 200 205Ile Ser Ile Asn Tyr Arg Thr Glu Ile
Asp Lys Pro Cys Gln His His 210 215 220His His His
His225121221PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 121Val Ser Asp Val Pro Arg Asp Leu
Glu Val Val Ala Ala Thr Pro Thr1 5 10 15Ser Leu Leu Ile Ser Trp His
Glu Arg Asp Gly Ser Arg Gln Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40 45Thr Val Pro Gly Gly
Val Arg Thr Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr
Thr Ile Thr Val Tyr Ala Val Thr Asp Tyr Phe Asn65 70 75 80Pro Thr
Thr His Glu Tyr Ile Tyr Gln Thr Thr Pro Ile Ser Ile Asn 85 90 95Tyr
Arg Thr Glu Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala 100 105
110Pro Ala Pro Ala Pro Ala Val Ser Asp Val Pro Arg Asp Leu Glu Val
115 120 125Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Ser Ala
Arg Leu 130 135 140Lys Val Ala Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn145 150 155 160Ser Pro Val Gln Glu Phe Thr Val Pro
Lys Asn Val Tyr Thr Ala Thr 165 170 175Ile Ser Gly Leu Lys Pro Gly
Val Asp Tyr Thr Ile Thr Val Tyr Ala 180 185 190Val Thr Arg Phe Arg
Asp Tyr Gln Pro Ile Ser Ile Asn Tyr Arg Thr 195 200 205Glu Ile Asp
Lys Pro Cys Gln His His His His His His 210 215
220122213PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 122Val Ser Asp Val Pro Arg Asp Leu Glu Val
Val Ala Ala Thr Pro Thr1 5 10 15Ser Leu Leu Ile Ser Trp Ser Ala Arg
Leu Lys Val Ala Arg Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly
Gly Asn Ser Pro Val Gln Glu Phe 35 40 45Thr Val Pro Lys Asn Val Tyr
Thr Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile
Thr Val Tyr Ala Val Thr Arg Phe Arg Asp65 70 75 80Tyr Gln Pro Ile
Ser Ile Asn Tyr Arg Thr Glu Ile Asp Lys Gly Ser 85 90 95Gly Ser Gly
Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser 100 105 110Gly
Ser Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr 115 120
125Pro Thr Ser Leu Leu Ile Ser Trp His Glu Arg Asp Gly Ser Arg Gln
130 135 140Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln145 150 155 160Glu Phe Thr Val Pro Gly Gly Val Arg Thr Ala
Thr Ile Ser Gly Leu 165 170 175Lys Pro Gly Val Asp Tyr Thr Ile Thr
Val Tyr Ala Val Thr Asp Tyr 180 185 190Phe Asn Pro Thr Thr His Glu
Tyr Ile Tyr Gln Thr Thr Pro Ile Ser 195 200 205Ile Asn Tyr Arg Thr
210123220PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 123Val Ser Asp Val Pro Arg Asp Leu Glu Val
Val Ala Ala Thr Pro Thr1 5 10 15Ser Leu Leu Ile Ser Trp Ser Ala Arg
Leu Lys Val Ala Arg Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly
Gly Asn Ser Pro Val Gln Glu Phe 35 40 45Thr Val Pro Lys Asn Val Tyr
Thr Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile
Thr Val Tyr Ala Val Thr Arg Phe Arg Asp65 70 75 80Tyr Gln Pro Ile
Ser Ile Asn Tyr Arg Thr Glu Ile Asp Lys Gly Ser 85 90 95Gly Ser Gly
Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser 100 105 110Gly
Ser Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr 115 120
125Pro Thr Ser Leu Leu Ile Ser Trp His Glu Arg Asp Gly Ser Arg Gln
130 135 140Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln145 150 155 160Glu Phe Thr Val Pro Gly Gly Val Arg Thr Ala
Thr Ile Ser Gly Leu 165 170 175Lys Pro Gly Val Asp Tyr Thr Ile Thr
Val Tyr Ala Val Thr Asp Tyr 180 185 190Phe Asn Pro Thr Thr His Glu
Tyr Ile Tyr Gln Thr Thr Pro Ile Ser 195 200 205Ile Asn Tyr Arg Thr
Glu Ile Asp Lys Pro Xaa Gln 210 215 220124228PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
124Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1
5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Ser Ala Arg Leu Lys Val Ala
Arg 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln 35 40 45Glu Phe Thr Val Pro Lys Asn Val Tyr Thr Ala Thr Ile
Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala
Val Thr Arg Phe65 70 75 80Arg Asp Tyr Gln Pro Ile Ser Ile Asn Tyr
Arg Thr Glu Ile Asp Lys 85 90 95Gly Ser Gly Ser Gly Ser Gly Ser Gly
Ser Gly Ser Gly Ser Gly Ser 100 105 110Gly Ser Gly Ser Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ala 115 120 125Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp His Glu Arg Asp Gly Ser 130 135 140Arg Gln Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro145 150 155
160Val Gln Glu Phe Thr Val Pro Gly Gly Val Arg Thr Ala Thr Ile Ser
165 170 175Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala
Val Thr 180 185 190Asp Tyr Phe Asn Pro Thr Thr His Glu Tyr Ile Tyr
Gln Thr Thr Pro 195 200 205Ile Ser Ile Asn Tyr Arg Thr Glu Ile Asp
Lys Pro Cys Gln His His 210 215 220His His His
His225125215PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 125Val Ser Asp Val Pro Arg Asp Leu
Glu Val Val Ala Ala Thr Pro Thr1 5 10 15Ser Leu Leu Ile Ser Trp Ser
Ala Arg Leu Lys Val Ala Arg Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40 45Thr Val Pro Lys Asn
Val Tyr Thr Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr
Thr Ile Thr Val Tyr Ala Val Thr Arg Phe Arg Asp65 70 75 80Tyr Gln
Pro Ile Ser Ile Asn Tyr Arg Thr Glu Pro Ala Pro Ala Pro 85 90 95Ala
Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Val Ser Asp 100 105
110Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu
115 120 125Ile Ser Trp His Glu Arg Asp Gly Ser Arg Gln Tyr Tyr Arg
Ile Thr 130 135 140Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu
Phe Thr Val Pro145 150 155 160Gly Gly Val Arg Thr Ala Thr Ile Ser
Gly Leu Lys Pro Gly Val Asp 165 170 175Tyr Thr Ile Thr Val Tyr Ala
Val Thr Asp Tyr Phe Asn Pro Thr Thr 180 185 190His Glu Tyr Ile Tyr
Gln Thr Thr Pro Ile Ser Ile Asn Tyr Arg Thr 195 200 205Glu Ile Asp
Lys Pro Xaa Gln 210 215126213PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 126Val Ser Asp Val Pro
Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr1 5 10 15Ser Leu Leu Ile
Ser Trp Trp Ala Pro Val Asp Arg Tyr Gln Tyr Tyr 20 25 30Arg Ile Thr
Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40 45Thr Val
Pro Arg Asp Val Tyr Thr Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly
Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Asp Tyr Lys Pro65 70 75
80His Ala Asp Gly Pro His Thr Tyr His Glu Ser Pro Ile Ser Ile Asn
85 90 95Tyr Arg Thr Glu Ile Asp Lys Gly Ser Gly Ser Gly Ser Gly Ser
Gly 100 105 110Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Val Ser
Asp Val Pro 115 120 125Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr
Ser Leu Leu Ile Ser 130 135 140Trp Ser Ala Arg Leu Lys Val Ala Arg
Tyr Tyr Arg Ile Thr Tyr Gly145 150 155 160Glu Thr Gly Gly Asn Ser
Pro Val Gln Glu Phe Thr Val Pro Lys Asn 165 170 175Val Tyr Thr Ala
Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr 180 185 190Ile Thr
Val Tyr Ala Val Thr Arg Phe Arg Asp Tyr Gln Pro Ile Ser 195 200
205Ile Asn Tyr Arg Thr 210127220PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 127Val Ser Asp Val Pro
Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr1 5 10 15Ser Leu Leu Ile
Ser Trp Trp Ala Pro Val Asp Arg Tyr Gln Tyr Tyr 20 25 30Arg Ile Thr
Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40 45Thr Val
Pro Arg Asp Val Tyr Thr Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly
Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Asp Tyr Lys Pro65 70 75
80His Ala Asp Gly Pro His Thr Tyr His Glu Ser Pro Ile Ser Ile Asn
85 90 95Tyr Arg Thr Glu Ile Asp Lys Gly Ser Gly Ser Gly Ser Gly Ser
Gly 100 105 110Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Val Ser
Asp Val Pro 115 120 125Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr
Ser Leu Leu Ile Ser 130 135 140Trp Ser Ala Arg Leu Lys Val Ala Arg
Tyr Tyr Arg Ile Thr Tyr Gly145 150 155 160Glu Thr Gly Gly Asn Ser
Pro Val Gln Glu Phe Thr Val Pro Lys Asn 165 170 175Val Tyr Thr Ala
Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr 180 185 190Ile Thr
Val Tyr Ala Val Thr Arg Phe Arg Asp Tyr Gln Pro Ile Ser 195 200
205Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Xaa Gln 210 215
220128228PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 128Met Gly Val Ser Asp Val Pro Arg Asp Leu
Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Trp
Ala Pro Val Asp Arg Tyr Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr Val Pro Arg Asp
Val Tyr Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr
Thr Ile Thr Val Tyr Ala Val Thr Asp Tyr65 70 75 80Lys Pro His Ala
Asp Gly Pro His Thr Tyr His Glu Ser Pro Ile Ser 85 90 95Ile Asn Tyr
Arg Thr Glu Ile Asp Lys Gly Ser Gly Ser Gly Ser Gly 100 105 110Ser
Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Val Ser Asp 115 120
125Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu
130 135 140Ile Ser Trp Ser Ala Arg Leu Lys Val Ala Arg Tyr Tyr Arg
Ile Thr145 150 155 160Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro 165 170 175Lys Asn Val Tyr Thr Ala Thr Ile Ser
Gly Leu Lys Pro Gly Val Asp 180 185 190Tyr Thr Ile Thr Val Tyr Ala
Val Thr Arg Phe Arg Asp Tyr Gln Pro 195 200 205Ile Ser Ile Asn Tyr
Arg Thr Glu Ile Asp Lys Pro Cys Gln His His 210 215 220His His His
His225129215PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 129Val Ser Asp Val Pro Arg Asp Leu
Glu Val Val Ala Ala Thr Pro Thr1 5 10 15Ser Leu Leu Ile Ser Trp Trp
Ala Pro Val Asp Arg Tyr Gln Tyr Tyr 20 25 30Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40 45Thr Val Pro Arg Asp
Val Tyr Thr Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly Val Asp Tyr
Thr Ile Thr Val Tyr Ala Val Thr Asp Tyr Lys Pro65 70 75 80His Ala
Asp Gly Pro His Thr Tyr His Glu Ser Pro Ile Ser Ile Asn 85 90 95Tyr
Arg Thr Glu Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala 100 105
110Pro Ala Pro Ala Pro Ala Val Ser Asp Val Pro Arg Asp Leu Glu Val
115 120 125Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Ser Ala
Arg Leu 130 135 140Lys Val Ala Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn145 150 155 160Ser Pro Val Gln Glu Phe Thr Val Pro
Lys Asn Val Tyr Thr Ala Thr 165 170 175Ile Ser Gly Leu Lys Pro Gly
Val Asp Tyr Thr Ile Thr Val Tyr Ala 180 185 190Val Thr Arg Phe Arg
Asp Tyr Gln Pro Ile Ser Ile Asn Tyr Arg Thr 195 200 205Glu Ile Asp
Lys Pro Xaa Gln 210 215130213PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 130Val Ser Asp Val Pro
Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr1 5 10 15Ser Leu Leu Ile
Ser Trp Ser Ala Arg Leu Lys Val Ala Arg Tyr Tyr 20 25 30Arg Ile Thr
Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40 45Thr Val
Pro Lys Asn Val Tyr Thr Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly
Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Arg Phe Arg Asp65 70 75
80Tyr Gln Pro Ile Ser Ile Asn Tyr Arg Thr Glu Ile Asp Lys Gly Ser
85 90 95Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly
Ser 100 105 110Gly Ser Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 115 120 125Pro Thr Ser Leu Leu Ile Ser Trp Trp Ala Pro
Val Asp Arg Tyr Gln 130 135 140Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln145 150 155 160Glu Phe Thr Val Pro Arg
Asp Val Tyr Thr Ala Thr Ile Ser Gly Leu 165 170 175Lys Pro Gly Val
Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Asp Tyr 180 185 190Lys Pro
His Ala Asp Gly Pro His Thr Tyr His Glu Ser Pro Ile Ser 195 200
205Ile Asn Tyr Arg Thr 210131220PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
131Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr1
5 10 15Ser Leu Leu Ile Ser Trp Ser Ala Arg Leu Lys Val Ala Arg Tyr
Tyr 20 25 30Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln
Glu Phe 35 40 45Thr Val Pro Lys Asn Val Tyr Thr Ala Thr Ile Ser Gly
Leu Lys Pro 50 55 60Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr
Arg Phe Arg Asp65 70 75 80Tyr Gln Pro Ile Ser Ile Asn Tyr Arg Thr
Glu Ile Asp Lys Gly Ser 85 90 95Gly Ser Gly Ser Gly Ser Gly Ser Gly
Ser Gly Ser Gly Ser Gly Ser 100 105 110Gly Ser Val Ser Asp Val Pro
Arg Asp Leu Glu Val Val Ala Ala Thr 115 120 125Pro Thr Ser Leu Leu
Ile Ser Trp Trp Ala Pro Val Asp Arg Tyr Gln 130 135 140Tyr Tyr Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln145 150 155
160Glu Phe Thr Val Pro Arg Asp Val Tyr Thr Ala Thr Ile Ser Gly Leu
165 170 175Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr
Asp Tyr 180 185 190Lys Pro His Ala Asp Gly Pro His Thr Tyr His Glu
Ser Pro Ile Ser 195 200 205Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro
Xaa Gln 210 215 220132228PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 132Met Gly Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu
Leu Ile Ser Trp Ser Ala Arg Leu Lys Val Ala Arg 20 25 30Tyr Tyr Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe
Thr Val Pro Lys Asn Val Tyr Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys
Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Arg Phe65 70 75
80Arg Asp Tyr Gln Pro Ile Ser Ile Asn Tyr Arg Thr Glu Ile Asp Lys
85 90 95Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly
Ser 100 105 110Gly Ser Gly Ser Val Ser Asp Val Pro Arg Asp Leu Glu
Val Val Ala 115 120 125Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Trp
Ala Pro Val Asp Arg 130 135 140Tyr Gln Tyr Tyr Arg Ile Thr Tyr Gly
Glu Thr Gly Gly Asn Ser Pro145 150 155 160Val Gln Glu Phe Thr Val
Pro Arg Asp Val Tyr Thr Ala Thr Ile Ser 165 170 175Gly Leu Lys Pro
Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr 180 185 190Asp Tyr
Lys Pro His Ala Asp Gly Pro His Thr Tyr His Glu Ser Pro 195 200
205Ile Ser Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Cys Gln His His
210 215 220His His His His225133217PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
133Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1
5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Ser Ala Arg Leu Lys Val Ala
Arg 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln 35 40 45Glu Phe Thr Val Pro Lys Asn Val Tyr Thr Ala Thr Ile
Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala
Val Thr Arg Phe65 70 75 80Arg Asp Tyr Gln Pro Ile Ser Ile Asn Tyr
Arg Thr Glu Pro Ala Pro 85 90 95Ala Pro Ala Pro Ala Pro Ala Pro Ala
Pro Ala Pro Ala Pro Ala Val 100 105 110Ser Asp Val Pro Arg Asp Leu
Glu Val Val Ala Ala Thr Pro Thr Ser 115 120 125Leu Leu Ile Ser Trp
Trp Ala Pro Val Asp Arg Tyr Gln Tyr Tyr Arg 130 135 140Ile Thr Tyr
Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe Thr145 150 155
160Val Pro Arg Asp Val Tyr Thr Ala Thr Ile Ser Gly Leu Lys Pro Gly
165 170 175Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Asp Tyr Lys
Pro His 180 185 190Ala Asp Gly Pro His Thr Tyr His Glu Ser Pro Ile
Ser Ile Asn Tyr 195 200 205Arg Thr Glu Ile Asp Lys Pro Xaa Gln 210
21513418PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 134Xaa Xaa Xaa Xaa Xaa His Glu Arg Asp Gly Ser
Arg Gln Xaa Xaa Xaa1 5 10 15Xaa Xaa13514PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 135Xaa
Xaa Xaa Xaa Xaa Gly Gly Val Arg Xaa Xaa Xaa Xaa Xaa1 5
1013625PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 136Xaa Xaa Xaa Xaa Xaa Asp Tyr Phe Asn Pro Thr
Thr His Glu Tyr Ile1 5 10 15Tyr Gln Thr Thr Xaa Xaa Xaa Xaa Xaa 20
2513718PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 137Xaa Xaa Xaa Xaa Xaa Trp Ala Pro Val Asp Arg
Tyr Gln Xaa Xaa Xaa1 5 10 15Xaa Xaa13814PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 138Xaa
Xaa Xaa Xaa Xaa Arg Asp Val Tyr Xaa Xaa Xaa Xaa Xaa1 5
1013925PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 139Xaa Xaa Xaa Xaa Xaa Asp Tyr Lys Pro His Ala
Asp Gly Pro His Thr1 5 10 15Tyr His Glu Ser Xaa Xaa Xaa Xaa Xaa 20
25140114PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 140Met Gly Val Ser Asp Val Pro Arg Asp Leu
Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Thr
Gln Gly Ser Thr His Tyr Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr Val Pro Gly Met
Val Tyr Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr
Thr Ile Thr Val Tyr Ala Val Thr Asp Tyr65 70 75 80Phe Asp Arg Ser
Thr His Glu Tyr Lys Tyr Arg Thr Thr Pro Ile Ser 85 90 95Ile Asn Tyr
Arg Thr Glu Ile Asp Lys Pro Ser Gln His His His His 100 105 110His
His14191PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 141Glu Val Val Ala Ala Thr Pro Thr Ser Leu
Leu Ile Ser Trp Thr Gln1 5 10 15Gly Ser Thr His Tyr Gln Tyr Tyr Arg
Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe
Thr Val Pro Gly Met Val Tyr Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys
Pro Gly Val Asp Tyr Thr Ile Thr Val 50 55 60Tyr Ala Val Thr Asp Tyr
Phe Asp Arg Ser Thr His Glu Tyr Lys Tyr65 70 75 80Arg Thr Thr Pro
Ile Ser Ile Asn Tyr Arg Thr 85 90142108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
142Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Glu Val Val Ala Ala Thr1
5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Thr Gln Gly Ser Thr His Tyr
Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln 35 40 45Glu Phe Thr Val Pro Gly Met Val Tyr Thr Ala Thr Ile
Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala
Val Thr Asp Tyr65 70 75 80Phe Asp Arg Ser Thr His Glu Tyr Lys Tyr
Arg Thr Thr Pro Ile Ser 85 90 95Ile Asn Tyr Arg Thr Xaa Xaa Xaa Xaa
Xaa Xaa Xaa 100 10514310PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 143Ser Trp Thr Gln Gly Ser
Thr His Tyr Gln1 5 101446PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 144Pro Gly Met Val Tyr Thr1
514517PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 145Thr Asp Tyr Phe Asp Arg Ser Thr His Glu Tyr
Lys Tyr Arg Thr Thr1 5 10 15Pro14618PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 146Xaa
Xaa Xaa Xaa Xaa Thr Gln Gly Ser Thr His Tyr Gln Xaa Xaa Xaa1 5 10
15Xaa Xaa14714PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 147Xaa Xaa Xaa Xaa Xaa Gly Met Val Tyr
Xaa Xaa Xaa Xaa Xaa1 5 1014825PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 148Xaa Xaa Xaa Xaa Xaa Asp
Tyr Phe Asp Arg Ser Thr His Glu Tyr Lys1 5 10 15Tyr Arg Thr Thr Xaa
Xaa Xaa Xaa Xaa 20 25149228PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 149Met Gly Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu
Leu Ile Ser Trp Thr Gln Gly Ser Thr His Tyr Gln 20 25 30Tyr Tyr Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe
Thr Val Pro Gly Met Val Tyr Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys
Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Asp Tyr65 70 75
80Phe Asp Arg Ser Thr His Glu Tyr Lys Tyr Arg Thr Thr Pro Ile Ser
85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys Gly Ser Gly Ser Gly Ser
Gly 100 105 110Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser
Val Ser Asp 115 120 125Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr
Pro Thr Ser Leu Leu 130 135 140Ile Ser Trp Ser Ala Arg Leu Lys Val
Ala Arg Tyr Tyr Arg Ile Thr145 150 155 160Tyr Gly Glu Thr Gly Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro 165 170 175Lys Asn Val Tyr
Thr Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp 180 185 190Tyr Thr
Ile Thr Val Tyr Ala Val Thr Arg Phe Arg Asp Tyr Gln Pro 195 200
205Ile Ser Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Cys Gln His His
210 215 220His His His His225150222PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
150Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Glu Val Val Ala Ala Thr1
5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Thr Gln Gly Ser Thr His Tyr
Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln 35 40 45Glu Phe Thr Val Pro Gly Met Val Tyr Thr Ala Thr Ile
Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala
Val Thr Asp Tyr65 70 75 80Phe Asp Arg Ser Thr His Glu Tyr Lys Tyr
Arg Thr Thr Pro Ile Ser 85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys
Gly Ser Gly Ser Gly Ser Gly 100 105 110Ser Gly Ser Gly Ser Gly Ser
Gly Ser Gly Ser Gly Ser Val Ser Asp 115 120 125Val Pro Arg Asp Leu
Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu 130 135 140Ile Ser Trp
Ser Ala Arg Leu Lys Val Ala Arg Tyr Tyr Arg Ile Thr145 150 155
160Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro
165 170 175Lys Asn Val Tyr Thr Ala Thr Ile Ser Gly Leu Lys Pro Gly
Val Asp 180 185 190Tyr Thr Ile Thr Val Tyr Ala Val Thr Arg Phe Arg
Asp Tyr Gln Pro 195 200 205Ile Ser Ile Asn Tyr Arg Thr Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 210 215 220151217PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 151Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu
Leu Ile Ser Trp Thr Gln Gly Ser Thr His Tyr Gln 20 25 30Tyr Tyr Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe
Thr Val Pro Gly Met Val Tyr Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys
Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Asp Tyr65 70 75
80Phe Asp Arg Ser Thr His Glu Tyr Lys Tyr Arg Thr Thr Pro Ile Ser
85 90 95Ile Asn Tyr Arg Thr Glu Pro Ala Pro Ala Pro Ala Pro Ala Pro
Ala 100 105 110Pro Ala Pro Ala Pro Ala Pro Ala Val Ser Asp Val Pro
Arg Asp Leu 115 120 125Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu
Ile Ser Trp Ser Ala 130 135 140Arg Leu Lys Val Ala Arg Tyr Tyr Arg
Ile Thr Tyr Gly Glu Thr Gly145 150 155 160Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Lys Asn Val Tyr Thr 165 170 175Ala Thr Ile Ser
Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Val 180 185 190Tyr Ala
Val Thr Arg Phe Arg Asp Tyr Gln Pro Ile Ser Ile Asn Tyr 195 200
205Arg Thr Xaa Xaa Xaa Xaa Xaa Xaa Xaa 210 215152228PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
152Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1
5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Ser Ala Arg Leu Lys Val Ala
Arg 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln 35 40 45Glu Phe Thr Val Pro Lys Asn Val Tyr Thr Ala Thr Ile
Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala
Val Thr Arg Phe65 70 75 80Arg Asp Tyr Gln Pro Ile Ser Ile Asn Tyr
Arg Thr Glu Ile Asp Lys 85 90 95Gly Ser Gly Ser Gly Ser Gly Ser Gly
Ser Gly Ser Gly Ser Gly Ser 100 105 110Gly Ser Gly Ser Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ala 115 120 125Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Thr Gln Gly Ser Thr His 130 135 140Tyr Gln Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro145 150 155
160Val Gln Glu Phe Thr Val Pro Gly Met Val Tyr Thr Ala Thr Ile Ser
165 170 175Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala
Val Thr 180 185 190Asp Tyr Phe Asp Arg Ser Thr His Glu Tyr Lys Tyr
Arg Thr Thr Pro 195 200 205Ile Ser Ile Asn Tyr Arg Thr Glu Ile Asp
Lys Pro Cys Gln His His 210 215 220His His His
His225153222PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 153Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu Leu Ile Ser
Trp Ser Ala Arg Leu Lys Val Ala Arg 20 25 30Tyr Tyr Arg Ile Thr Tyr
Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr Val Pro
Lys Asn Val Tyr Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro Gly Val
Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Arg Phe65 70 75 80Arg Asp
Tyr Gln Pro Ile Ser Ile Asn Tyr Arg Thr Glu Ile Asp Lys 85 90 95Gly
Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser 100 105
110Gly Ser Gly Ser Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala
115 120 125Ala Thr
Pro Thr Ser Leu Leu Ile Ser Trp Thr Gln Gly Ser Thr His 130 135
140Tyr Gln Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser
Pro145 150 155 160Val Gln Glu Phe Thr Val Pro Gly Met Val Tyr Thr
Ala Thr Ile Ser 165 170 175Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile
Thr Val Tyr Ala Val Thr 180 185 190Asp Tyr Phe Asp Arg Ser Thr His
Glu Tyr Lys Tyr Arg Thr Thr Pro 195 200 205Ile Ser Ile Asn Tyr Arg
Thr Xaa Xaa Xaa Xaa Xaa Xaa Xaa 210 215 220154217PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
154Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Glu Val Val Ala Ala Thr1
5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Ser Ala Arg Leu Lys Val Ala
Arg 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln 35 40 45Glu Phe Thr Val Pro Lys Asn Val Tyr Thr Ala Thr Ile
Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala
Val Thr Arg Phe65 70 75 80Arg Asp Tyr Gln Pro Ile Ser Ile Asn Tyr
Arg Thr Glu Pro Ala Pro 85 90 95Ala Pro Ala Pro Ala Pro Ala Pro Ala
Pro Ala Pro Ala Pro Ala Val 100 105 110Ser Asp Val Pro Arg Asp Leu
Glu Val Val Ala Ala Thr Pro Thr Ser 115 120 125Leu Leu Ile Ser Trp
Thr Gln Gly Ser Thr His Tyr Gln Tyr Tyr Arg 130 135 140Ile Thr Tyr
Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe Thr145 150 155
160Val Pro Gly Met Val Tyr Thr Ala Thr Ile Ser Gly Leu Lys Pro Gly
165 170 175Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Asp Tyr Phe
Asp Arg 180 185 190Ser Thr His Glu Tyr Lys Tyr Arg Thr Thr Pro Ile
Ser Ile Asn Tyr 195 200 205Arg Thr Xaa Xaa Xaa Xaa Xaa Xaa Xaa 210
215155114PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 155Met Gly Val Ser Asp Val Pro Arg Asp Leu
Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Tyr
Trp Glu Gly Leu Pro Tyr Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr Val Pro Arg Asp
Val Asn Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr
Thr Ile Thr Val Tyr Ala Val Thr Asp Trp65 70 75 80Tyr Asn Pro Asp
Thr His Glu Tyr Ile Tyr His Thr Ile Pro Ile Ser 85 90 95Ile Asn Tyr
Arg Thr Glu Ile Asp Lys Pro Ser Gln His His His His 100 105 110His
His15691PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 156Glu Val Val Ala Ala Thr Pro Thr Ser Leu
Leu Ile Ser Trp Tyr Trp1 5 10 15Glu Gly Leu Pro Tyr Gln Tyr Tyr Arg
Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe
Thr Val Pro Arg Asp Val Asn Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys
Pro Gly Val Asp Tyr Thr Ile Thr Val 50 55 60Tyr Ala Val Thr Asp Trp
Tyr Asn Pro Asp Thr His Glu Tyr Ile Tyr65 70 75 80His Thr Ile Pro
Ile Ser Ile Asn Tyr Arg Thr 85 90157108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
157Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Glu Val Val Ala Ala Thr1
5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Tyr Trp Glu Gly Leu Pro Tyr
Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln 35 40 45Glu Phe Thr Val Pro Arg Asp Val Asn Thr Ala Thr Ile
Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala
Val Thr Asp Trp65 70 75 80Tyr Asn Pro Asp Thr His Glu Tyr Ile Tyr
His Thr Ile Pro Ile Ser 85 90 95Ile Asn Tyr Arg Thr Xaa Xaa Xaa Xaa
Xaa Xaa Xaa 100 10515810PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 158 Ser Trp Tyr Trp Glu Gly
Leu Pro Tyr Gln1 5 10 1596PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 159Pro Arg Asp Val Asn Thr1
516017PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 160Thr Asp Trp Tyr Asn Pro Asp Thr His Glu Tyr
Ile Tyr His Thr Ile1 5 10 15Pro16118PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 161Xaa
Xaa Xaa Xaa Xaa Tyr Trp Glu Gly Leu Pro Tyr Gln Xaa Xaa Xaa1 5 10
15Xaa Xaa16214PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 162Xaa Xaa Xaa Xaa Xaa Arg Asp Val Asn
Xaa Xaa Xaa Xaa Xaa1 5 1016325PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 163Xaa Xaa Xaa Xaa Xaa Asp
Trp Tyr Asn Pro Asp Thr His Glu Tyr Ile1 5 10 15Tyr His Thr Ile Xaa
Xaa Xaa Xaa Xaa 20 25164228PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 164Met Gly Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu
Leu Ile Ser Trp Tyr Trp Glu Gly Leu Pro Tyr Gln 20 25 30Tyr Tyr Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe
Thr Val Pro Arg Asp Val Asn Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys
Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Asp Trp65 70 75
80Tyr Asn Pro Asp Thr His Glu Tyr Ile Tyr His Thr Ile Pro Ile Ser
85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys Gly Ser Gly Ser Gly Ser
Gly 100 105 110Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser
Val Ser Asp 115 120 125Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr
Pro Thr Ser Leu Leu 130 135 140Ile Ser Trp Ser Ala Arg Leu Lys Val
Ala Arg Tyr Tyr Arg Ile Thr145 150 155 160Tyr Gly Glu Thr Gly Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro 165 170 175Lys Asn Val Tyr
Thr Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp 180 185 190Tyr Thr
Ile Thr Val Tyr Ala Val Thr Arg Phe Arg Asp Tyr Gln Pro 195 200
205Ile Ser Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Cys Gln His His
210 215 220His His His His225165222PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
165Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Glu Val Val Ala Ala Thr1
5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Tyr Trp Glu Gly Leu Pro Tyr
Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln 35 40 45Glu Phe Thr Val Pro Arg Asp Val Asn Thr Ala Thr Ile
Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala
Val Thr Asp Trp65 70 75 80Tyr Asn Pro Asp Thr His Glu Tyr Ile Tyr
His Thr Ile Pro Ile Ser 85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys
Gly Ser Gly Ser Gly Ser Gly 100 105 110Ser Gly Ser Gly Ser Gly Ser
Gly Ser Gly Ser Gly Ser Val Ser Asp 115 120 125Val Pro Arg Asp Leu
Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu 130 135 140Ile Ser Trp
Ser Ala Arg Leu Lys Val Ala Arg Tyr Tyr Arg Ile Thr145 150 155
160Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro
165 170 175Lys Asn Val Tyr Thr Ala Thr Ile Ser Gly Leu Lys Pro Gly
Val Asp 180 185 190Tyr Thr Ile Thr Val Tyr Ala Val Thr Arg Phe Arg
Asp Tyr Gln Pro 195 200 205Ile Ser Ile Asn Tyr Arg Thr Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 210 215 220166217PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 166Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu
Leu Ile Ser Trp Tyr Trp Glu Gly Leu Pro Tyr Gln 20 25 30Tyr Tyr Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe
Thr Val Pro Arg Asp Val Asn Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys
Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Asp Trp65 70 75
80Tyr Asn Pro Asp Thr His Glu Tyr Ile Tyr His Thr Ile Pro Ile Ser
85 90 95Ile Asn Tyr Arg Thr Glu Pro Ala Pro Ala Pro Ala Pro Ala Pro
Ala 100 105 110Pro Ala Pro Ala Pro Ala Pro Ala Val Ser Asp Val Pro
Arg Asp Leu 115 120 125Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu
Ile Ser Trp Ser Ala 130 135 140Arg Leu Lys Val Ala Arg Tyr Tyr Arg
Ile Thr Tyr Gly Glu Thr Gly145 150 155 160Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro Lys Asn Val Tyr Thr 165 170 175Ala Thr Ile Ser
Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Val 180 185 190Tyr Ala
Val Thr Arg Phe Arg Asp Tyr Gln Pro Ile Ser Ile Asn Tyr 195 200
205Arg Thr Xaa Xaa Xaa Xaa Xaa Xaa Xaa 210 215167228PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
167Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1
5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Ser Ala Arg Leu Lys Val Ala
Arg 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln 35 40 45Glu Phe Thr Val Pro Lys Asn Val Tyr Thr Ala Thr Ile
Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala
Val Thr Arg Phe65 70 75 80Arg Asp Tyr Gln Pro Ile Ser Ile Asn Tyr
Arg Thr Glu Ile Asp Lys 85 90 95Gly Ser Gly Ser Gly Ser Gly Ser Gly
Ser Gly Ser Gly Ser Gly Ser 100 105 110Gly Ser Gly Ser Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ala 115 120 125Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Tyr Trp Glu Gly Leu Pro 130 135 140Tyr Gln Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro145 150 155
160Val Gln Glu Phe Thr Val Pro Arg Asp Val Asn Thr Ala Thr Ile Ser
165 170 175Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala
Val Thr 180 185 190Asp Trp Tyr Asn Pro Asp Thr His Glu Tyr Ile Tyr
His Thr Ile Pro 195 200 205Ile Ser Ile Asn Tyr Arg Thr Glu Ile Asp
Lys Pro Cys Gln His His 210 215 220His His His
His225168222PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 168Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu Leu Ile Ser
Trp Ser Ala Arg Leu Lys Val Ala Arg 20 25 30Tyr Tyr Arg Ile Thr Tyr
Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr Val Pro
Lys Asn Val Tyr Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro Gly Val
Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Arg Phe65 70 75 80Arg Asp
Tyr Gln Pro Ile Ser Ile Asn Tyr Arg Thr Glu Ile Asp Lys 85 90 95Gly
Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser 100 105
110Gly Ser Gly Ser Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala
115 120 125Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Tyr Trp Glu Gly
Leu Pro 130 135 140Tyr Gln Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly
Gly Asn Ser Pro145 150 155 160Val Gln Glu Phe Thr Val Pro Arg Asp
Val Asn Thr Ala Thr Ile Ser 165 170 175Gly Leu Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Val Thr 180 185 190Asp Trp Tyr Asn Pro
Asp Thr His Glu Tyr Ile Tyr His Thr Ile Pro 195 200 205Ile Ser Ile
Asn Tyr Arg Thr Xaa Xaa Xaa Xaa Xaa Xaa Xaa 210 215
220169217PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 169Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Ser
Ala Arg Leu Lys Val Ala Arg 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr Val Pro Lys Asn
Val Tyr Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr
Thr Ile Thr Val Tyr Ala Val Thr Arg Phe65 70 75 80Arg Asp Tyr Gln
Pro Ile Ser Ile Asn Tyr Arg Thr Glu Pro Ala Pro 85 90 95Ala Pro Ala
Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Val 100 105 110Ser
Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr Ser 115 120
125Leu Leu Ile Ser Trp Tyr Trp Glu Gly Leu Pro Tyr Gln Tyr Tyr Arg
130 135 140Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu
Phe Thr145 150 155 160Val Pro Arg Asp Val Asn Thr Ala Thr Ile Ser
Gly Leu Lys Pro Gly 165 170 175Val Asp Tyr Thr Ile Thr Val Tyr Ala
Val Thr Asp Trp Tyr Asn Pro 180 185 190Asp Thr His Glu Tyr Ile Tyr
His Thr Ile Pro Ile Ser Ile Asn Tyr 195 200 205Arg Thr Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 210 215170114PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 170Met Gly Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu
Leu Ile Ser Trp Ala Ser Asn Arg Gly Thr Tyr Gln 20 25 30Tyr Tyr Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe
Thr Val Pro Gly Gly Val Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys
Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Asp Ala65 70 75
80Phe Asn Pro Thr Thr His Glu Tyr Asn Tyr Phe Thr Thr Pro Ile Ser
85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln His His His
His 100 105 110His His17191PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 171Glu Val Val Ala Ala
Thr Pro Thr Ser Leu Leu Ile Ser Trp Ala Ser1 5 10 15Asn Arg Gly Thr
Tyr Gln Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30Gly Asn Ser
Pro Val Gln Glu Phe Thr Val Pro Gly Gly Val Ser Thr 35 40 45Ala Thr
Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Val 50 55 60Tyr
Ala Val Thr Asp Ala Phe Asn Pro Thr Thr His Glu Tyr Asn Tyr65 70 75
80Phe Thr Thr Pro Ile Ser Ile Asn Tyr Arg Thr 85
90172108PRTArtificial SequenceDescription of Artificial
Sequence
Synthetic polypeptide 172Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Ala
Ser Asn Arg Gly Thr Tyr Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr Val Pro Gly Gly
Val Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr
Thr Ile Thr Val Tyr Ala Val Thr Asp Ala65 70 75 80Phe Asn Pro Thr
Thr His Glu Tyr Asn Tyr Phe Thr Thr Pro Ile Ser 85 90 95Ile Asn Tyr
Arg Thr Xaa Xaa Xaa Xaa Xaa Xaa Xaa 100 10517310PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 173Ser
Trp Ala Ser Asn Arg Gly Thr Tyr Gln1 5 101746PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 174Pro
Gly Gly Val Ser Thr1 517517PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 175Thr Asp Ala Phe Asn Pro
Thr Thr His Glu Tyr Asn Tyr Phe Thr Thr1 5 10
15Pro17618PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 176Xaa Xaa Xaa Xaa Xaa Ala Ser Asn Arg Gly Thr
Tyr Gln Xaa Xaa Xaa1 5 10 15Xaa Xaa17714PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 177Xaa
Xaa Xaa Xaa Xaa Gly Gly Val Ser Xaa Xaa Xaa Xaa Xaa1 5
1017825PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 178Xaa Xaa Xaa Xaa Xaa Asp Ala Phe Asn Pro Thr
Thr His Glu Tyr Asn1 5 10 15Tyr Phe Thr Thr Xaa Xaa Xaa Xaa Xaa 20
25179228PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 179Met Gly Val Ser Asp Val Pro Arg Asp Leu
Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Ala
Ser Asn Arg Gly Thr Tyr Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr Val Pro Gly Gly
Val Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr
Thr Ile Thr Val Tyr Ala Val Thr Asp Ala65 70 75 80Phe Asn Pro Thr
Thr His Glu Tyr Asn Tyr Phe Thr Thr Pro Ile Ser 85 90 95Ile Asn Tyr
Arg Thr Glu Ile Asp Lys Gly Ser Gly Ser Gly Ser Gly 100 105 110Ser
Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Val Ser Asp 115 120
125Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu
130 135 140Ile Ser Trp Ser Ala Arg Leu Lys Val Ala Arg Tyr Tyr Arg
Ile Thr145 150 155 160Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln
Glu Phe Thr Val Pro 165 170 175Lys Asn Val Tyr Thr Ala Thr Ile Ser
Gly Leu Lys Pro Gly Val Asp 180 185 190Tyr Thr Ile Thr Val Tyr Ala
Val Thr Arg Phe Arg Asp Tyr Gln Pro 195 200 205Ile Ser Ile Asn Tyr
Arg Thr Glu Ile Asp Lys Pro Cys Gln His His 210 215 220His His His
His225180222PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 180Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu Leu Ile Ser
Trp Ala Ser Asn Arg Gly Thr Tyr Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr
Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr Val Pro
Gly Gly Val Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro Gly Val
Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Asp Ala65 70 75 80Phe Asn
Pro Thr Thr His Glu Tyr Asn Tyr Phe Thr Thr Pro Ile Ser 85 90 95Ile
Asn Tyr Arg Thr Glu Ile Asp Lys Gly Ser Gly Ser Gly Ser Gly 100 105
110Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Val Ser Asp
115 120 125Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu 130 135 140Ile Ser Trp Ser Ala Arg Leu Lys Val Ala Arg Tyr
Tyr Arg Ile Thr145 150 155 160Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln Glu Phe Thr Val Pro 165 170 175Lys Asn Val Tyr Thr Ala Thr
Ile Ser Gly Leu Lys Pro Gly Val Asp 180 185 190Tyr Thr Ile Thr Val
Tyr Ala Val Thr Arg Phe Arg Asp Tyr Gln Pro 195 200 205Ile Ser Ile
Asn Tyr Arg Thr Xaa Xaa Xaa Xaa Xaa Xaa Xaa 210 215
220181217PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 181Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Ala
Ser Asn Arg Gly Thr Tyr Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr Val Pro Gly Gly
Val Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr
Thr Ile Thr Val Tyr Ala Val Thr Asp Ala65 70 75 80Phe Asn Pro Thr
Thr His Glu Tyr Asn Tyr Phe Thr Thr Pro Ile Ser 85 90 95Ile Asn Tyr
Arg Thr Glu Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala 100 105 110Pro
Ala Pro Ala Pro Ala Pro Ala Val Ser Asp Val Pro Arg Asp Leu 115 120
125Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Ser Ala
130 135 140Arg Leu Lys Val Ala Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly145 150 155 160Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro
Lys Asn Val Tyr Thr 165 170 175Ala Thr Ile Ser Gly Leu Lys Pro Gly
Val Asp Tyr Thr Ile Thr Val 180 185 190Tyr Ala Val Thr Arg Phe Arg
Asp Tyr Gln Pro Ile Ser Ile Asn Tyr 195 200 205Arg Thr Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 210 215182228PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 182Met Gly Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu
Leu Ile Ser Trp Ser Ala Arg Leu Lys Val Ala Arg 20 25 30Tyr Tyr Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe
Thr Val Pro Lys Asn Val Tyr Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys
Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Arg Phe65 70 75
80Arg Asp Tyr Gln Pro Ile Ser Ile Asn Tyr Arg Thr Glu Ile Asp Lys
85 90 95Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly
Ser 100 105 110Gly Ser Gly Ser Val Ser Asp Val Pro Arg Asp Leu Glu
Val Val Ala 115 120 125Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Ala
Ser Asn Arg Gly Thr 130 135 140Tyr Gln Tyr Tyr Arg Ile Thr Tyr Gly
Glu Thr Gly Gly Asn Ser Pro145 150 155 160Val Gln Glu Phe Thr Val
Pro Gly Gly Val Ser Thr Ala Thr Ile Ser 165 170 175Gly Leu Lys Pro
Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr 180 185 190Asp Ala
Phe Asn Pro Thr Thr His Glu Tyr Asn Tyr Phe Thr Thr Pro 195 200
205Ile Ser Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Cys Gln His His
210 215 220His His His His225183222PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
183Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Glu Val Val Ala Ala Thr1
5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Ser Ala Arg Leu Lys Val Ala
Arg 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln 35 40 45Glu Phe Thr Val Pro Lys Asn Val Tyr Thr Ala Thr Ile
Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala
Val Thr Arg Phe65 70 75 80Arg Asp Tyr Gln Pro Ile Ser Ile Asn Tyr
Arg Thr Glu Ile Asp Lys 85 90 95Gly Ser Gly Ser Gly Ser Gly Ser Gly
Ser Gly Ser Gly Ser Gly Ser 100 105 110Gly Ser Gly Ser Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ala 115 120 125Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Ala Ser Asn Arg Gly Thr 130 135 140Tyr Gln Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro145 150 155
160Val Gln Glu Phe Thr Val Pro Gly Gly Val Ser Thr Ala Thr Ile Ser
165 170 175Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala
Val Thr 180 185 190Asp Ala Phe Asn Pro Thr Thr His Glu Tyr Asn Tyr
Phe Thr Thr Pro 195 200 205Ile Ser Ile Asn Tyr Arg Thr Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 210 215 220184217PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 184Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu
Leu Ile Ser Trp Ser Ala Arg Leu Lys Val Ala Arg 20 25 30Tyr Tyr Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe
Thr Val Pro Lys Asn Val Tyr Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys
Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Arg Phe65 70 75
80Arg Asp Tyr Gln Pro Ile Ser Ile Asn Tyr Arg Thr Glu Pro Ala Pro
85 90 95Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala
Val 100 105 110Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr
Pro Thr Ser 115 120 125Leu Leu Ile Ser Trp Ala Ser Asn Arg Gly Thr
Tyr Gln Tyr Tyr Arg 130 135 140Ile Thr Tyr Gly Glu Thr Gly Gly Asn
Ser Pro Val Gln Glu Phe Thr145 150 155 160Val Pro Gly Gly Val Ser
Thr Ala Thr Ile Ser Gly Leu Lys Pro Gly 165 170 175Val Asp Tyr Thr
Ile Thr Val Tyr Ala Val Thr Asp Ala Phe Asn Pro 180 185 190Thr Thr
His Glu Tyr Asn Tyr Phe Thr Thr Pro Ile Ser Ile Asn Tyr 195 200
205Arg Thr Xaa Xaa Xaa Xaa Xaa Xaa Xaa 210 215185114PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
185Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1
5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro Thr Ser Arg Tyr
Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln 35 40 45Glu Phe Thr Val Pro Gly Gly Leu Ser Thr Ala Thr Ile
Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala
Val Thr Asp Tyr65 70 75 80Lys Pro His Ala Asp Gly Pro His Thr Tyr
His Glu Ser Pro Ile Ser 85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys
Pro Ser Gln His His His His 100 105 110His His18691PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
186Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala1
5 10 15Pro Thr Ser Arg Tyr Gln Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Gly Gly Leu
Ser Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr
Ile Thr Val 50 55 60Tyr Ala Val Thr Asp Tyr Lys Pro His Ala Asp Gly
Pro His Thr Tyr65 70 75 80His Glu Ser Pro Ile Ser Ile Asn Tyr Arg
Thr 85 90187108PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 187Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu Leu Ile Ser
Trp Asp Ala Pro Thr Ser Arg Tyr Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr
Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr Val Pro
Gly Gly Leu Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro Gly Val
Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Asp Tyr65 70 75 80Lys Pro
His Ala Asp Gly Pro His Thr Tyr His Glu Ser Pro Ile Ser 85 90 95Ile
Asn Tyr Arg Thr Xaa Xaa Xaa Xaa Xaa Xaa Xaa 100
10518810PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 188Ser Trp Asp Ala Pro Thr Ser Arg Tyr Gln1 5
101896PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 189Pro Gly Gly Leu Ser Thr1 519018PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 190Xaa
Xaa Xaa Xaa Xaa Asp Ala Pro Thr Ser Arg Tyr Gln Xaa Xaa Xaa1 5 10
15Xaa Xaa19114PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 191Xaa Xaa Xaa Xaa Xaa Gly Gly Leu Ser
Xaa Xaa Xaa Xaa Xaa1 5 10192228PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 192Met Gly Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu
Leu Ile Ser Trp Asp Ala Pro Thr Ser Arg Tyr Gln 20 25 30Tyr Tyr Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe
Thr Val Pro Gly Gly Leu Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys
Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Asp Tyr65 70 75
80Lys Pro His Ala Asp Gly Pro His Thr Tyr His Glu Ser Pro Ile Ser
85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys Gly Ser Gly Ser Gly Ser
Gly 100 105 110Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser
Val Ser Asp 115 120 125Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr
Pro Thr Ser Leu Leu 130 135 140Ile Ser Trp Ser Ala Arg Leu Lys Val
Ala Arg Tyr Tyr Arg Ile Thr145 150 155 160Tyr Gly Glu Thr Gly Gly
Asn Ser Pro Val Gln Glu Phe Thr Val Pro 165 170 175Lys Asn Val Tyr
Thr Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp 180 185 190Tyr Thr
Ile Thr Val Tyr Ala Val Thr Arg Phe Arg Asp Tyr Gln Pro 195 200
205Ile Ser Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Cys Gln His His
210 215 220His His His His225193222PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
193Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Glu Val Val Ala Ala Thr1
5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro Thr Ser Arg Tyr
Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln 35 40 45Glu Phe Thr Val Pro Gly Gly Leu Ser Thr Ala Thr Ile
Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala
Val Thr Asp Tyr65 70 75 80Lys Pro His Ala Asp Gly Pro His Thr Tyr
His Glu Ser Pro Ile Ser 85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys
Gly Ser Gly Ser Gly Ser Gly 100
105 110Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Val Ser
Asp 115 120 125Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr
Ser Leu Leu 130 135 140Ile Ser Trp Ser Ala Arg Leu Lys Val Ala Arg
Tyr Tyr Arg Ile Thr145 150 155 160Tyr Gly Glu Thr Gly Gly Asn Ser
Pro Val Gln Glu Phe Thr Val Pro 165 170 175Lys Asn Val Tyr Thr Ala
Thr Ile Ser Gly Leu Lys Pro Gly Val Asp 180 185 190Tyr Thr Ile Thr
Val Tyr Ala Val Thr Arg Phe Arg Asp Tyr Gln Pro 195 200 205Ile Ser
Ile Asn Tyr Arg Thr Xaa Xaa Xaa Xaa Xaa Xaa Xaa 210 215
220194217PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 194Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Asp
Ala Pro Thr Ser Arg Tyr Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr Val Pro Gly Gly
Leu Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr
Thr Ile Thr Val Tyr Ala Val Thr Asp Tyr65 70 75 80Lys Pro His Ala
Asp Gly Pro His Thr Tyr His Glu Ser Pro Ile Ser 85 90 95Ile Asn Tyr
Arg Thr Glu Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala 100 105 110Pro
Ala Pro Ala Pro Ala Pro Ala Val Ser Asp Val Pro Arg Asp Leu 115 120
125Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Ser Ala
130 135 140Arg Leu Lys Val Ala Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly145 150 155 160Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro
Lys Asn Val Tyr Thr 165 170 175Ala Thr Ile Ser Gly Leu Lys Pro Gly
Val Asp Tyr Thr Ile Thr Val 180 185 190Tyr Ala Val Thr Arg Phe Arg
Asp Tyr Gln Pro Ile Ser Ile Asn Tyr 195 200 205Arg Thr Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 210 215195228PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 195Met Gly Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu
Leu Ile Ser Trp Ser Ala Arg Leu Lys Val Ala Arg 20 25 30Tyr Tyr Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe
Thr Val Pro Lys Asn Val Tyr Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys
Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Arg Phe65 70 75
80Arg Asp Tyr Gln Pro Ile Ser Ile Asn Tyr Arg Thr Glu Ile Asp Lys
85 90 95Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly
Ser 100 105 110Gly Ser Gly Ser Val Ser Asp Val Pro Arg Asp Leu Glu
Val Val Ala 115 120 125Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Asp
Ala Pro Thr Ser Arg 130 135 140Tyr Gln Tyr Tyr Arg Ile Thr Tyr Gly
Glu Thr Gly Gly Asn Ser Pro145 150 155 160Val Gln Glu Phe Thr Val
Pro Gly Gly Leu Ser Thr Ala Thr Ile Ser 165 170 175Gly Leu Lys Pro
Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr 180 185 190Asp Tyr
Lys Pro His Ala Asp Gly Pro His Thr Tyr His Glu Ser Pro 195 200
205Ile Ser Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Cys Gln His His
210 215 220His His His His225196222PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
196Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Glu Val Val Ala Ala Thr1
5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Ser Ala Arg Leu Lys Val Ala
Arg 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln 35 40 45Glu Phe Thr Val Pro Lys Asn Val Tyr Thr Ala Thr Ile
Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala
Val Thr Arg Phe65 70 75 80Arg Asp Tyr Gln Pro Ile Ser Ile Asn Tyr
Arg Thr Glu Ile Asp Lys 85 90 95Gly Ser Gly Ser Gly Ser Gly Ser Gly
Ser Gly Ser Gly Ser Gly Ser 100 105 110Gly Ser Gly Ser Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ala 115 120 125Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Asp Ala Pro Thr Ser Arg 130 135 140Tyr Gln Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro145 150 155
160Val Gln Glu Phe Thr Val Pro Gly Gly Leu Ser Thr Ala Thr Ile Ser
165 170 175Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala
Val Thr 180 185 190Asp Tyr Lys Pro His Ala Asp Gly Pro His Thr Tyr
His Glu Ser Pro 195 200 205Ile Ser Ile Asn Tyr Arg Thr Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 210 215 220197217PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 197Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu
Leu Ile Ser Trp Ser Ala Arg Leu Lys Val Ala Arg 20 25 30Tyr Tyr Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe
Thr Val Pro Lys Asn Val Tyr Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys
Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Arg Phe65 70 75
80Arg Asp Tyr Gln Pro Ile Ser Ile Asn Tyr Arg Thr Glu Pro Ala Pro
85 90 95Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala
Val 100 105 110Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr
Pro Thr Ser 115 120 125Leu Leu Ile Ser Trp Asp Ala Pro Thr Ser Arg
Tyr Gln Tyr Tyr Arg 130 135 140Ile Thr Tyr Gly Glu Thr Gly Gly Asn
Ser Pro Val Gln Glu Phe Thr145 150 155 160Val Pro Gly Gly Leu Ser
Thr Ala Thr Ile Ser Gly Leu Lys Pro Gly 165 170 175Val Asp Tyr Thr
Ile Thr Val Tyr Ala Val Thr Asp Tyr Lys Pro His 180 185 190Ala Asp
Gly Pro His Thr Tyr His Glu Ser Pro Ile Ser Ile Asn Tyr 195 200
205Arg Thr Xaa Xaa Xaa Xaa Xaa Xaa Xaa 210 215198114PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
198Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1
5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Gly Ala Val Thr Tyr
Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln 35 40 45Glu Phe Thr Val Pro Gly Gly Val Arg Thr Ala Thr Ile
Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala
Val Thr Asp Tyr65 70 75 80Lys Pro His Ala Asp Gly Pro His Thr Tyr
His Glu Tyr Pro Ile Ser 85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys
Pro Ser Gln His His His His 100 105 110His His19991PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
199Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala1
5 10 15Gly Ala Val Thr Tyr Gln Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly 20 25 30Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Gly Gly Val
Arg Thr 35 40 45Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr
Ile Thr Val 50 55 60Tyr Ala Val Thr Asp Tyr Lys Pro His Ala Asp Gly
Pro His Thr Tyr65 70 75 80His Glu Tyr Pro Ile Ser Ile Asn Tyr Arg
Thr 85 90200108PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 200Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu Leu Ile Ser
Trp Asp Ala Gly Ala Val Thr Tyr Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr
Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr Val Pro
Gly Gly Val Arg Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro Gly Val
Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Asp Tyr65 70 75 80Lys Pro
His Ala Asp Gly Pro His Thr Tyr His Glu Tyr Pro Ile Ser 85 90 95Ile
Asn Tyr Arg Thr Xaa Xaa Xaa Xaa Xaa Xaa Xaa 100
10520110PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 201Ser Trp Asp Ala Gly Ala Val Thr Tyr Gln1 5
1020217PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 202Thr Asp Tyr Lys Pro His Ala Asp Gly Pro His
Thr Tyr His Glu Tyr1 5 10 15Pro20318PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 203Xaa
Xaa Xaa Xaa Xaa Asp Ala Gly Ala Val Thr Tyr Gln Xaa Xaa Xaa1 5 10
15Xaa Xaa20425PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 204Xaa Xaa Xaa Xaa Xaa Asp Tyr Lys Pro
His Ala Asp Gly Pro His Thr1 5 10 15Tyr His Glu Tyr Xaa Xaa Xaa Xaa
Xaa 20 25205228PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 205Met Gly Val Ser Asp Val Pro Arg
Asp Leu Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu Leu Ile Ser
Trp Asp Ala Gly Ala Val Thr Tyr Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr
Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr Val Pro
Gly Gly Val Arg Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro Gly Val
Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Asp Tyr65 70 75 80Lys Pro
His Ala Asp Gly Pro His Thr Tyr His Glu Tyr Pro Ile Ser 85 90 95Ile
Asn Tyr Arg Thr Glu Ile Asp Lys Gly Ser Gly Ser Gly Ser Gly 100 105
110Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Val Ser Asp
115 120 125Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu 130 135 140Ile Ser Trp Ser Ala Arg Leu Lys Val Ala Arg Tyr
Tyr Arg Ile Thr145 150 155 160Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln Glu Phe Thr Val Pro 165 170 175Lys Asn Val Tyr Thr Ala Thr
Ile Ser Gly Leu Lys Pro Gly Val Asp 180 185 190Tyr Thr Ile Thr Val
Tyr Ala Val Thr Arg Phe Arg Asp Tyr Gln Pro 195 200 205Ile Ser Ile
Asn Tyr Arg Thr Glu Ile Asp Lys Pro Cys Gln His His 210 215 220His
His His His225206222PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 206Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu Leu Ile Ser
Trp Asp Ala Gly Ala Val Thr Tyr Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr
Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr Val Pro
Gly Gly Val Arg Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro Gly Val
Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Asp Tyr65 70 75 80Lys Pro
His Ala Asp Gly Pro His Thr Tyr His Glu Tyr Pro Ile Ser 85 90 95Ile
Asn Tyr Arg Thr Glu Ile Asp Lys Gly Ser Gly Ser Gly Ser Gly 100 105
110Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Val Ser Asp
115 120 125Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr Ser
Leu Leu 130 135 140Ile Ser Trp Ser Ala Arg Leu Lys Val Ala Arg Tyr
Tyr Arg Ile Thr145 150 155 160Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln Glu Phe Thr Val Pro 165 170 175Lys Asn Val Tyr Thr Ala Thr
Ile Ser Gly Leu Lys Pro Gly Val Asp 180 185 190Tyr Thr Ile Thr Val
Tyr Ala Val Thr Arg Phe Arg Asp Tyr Gln Pro 195 200 205Ile Ser Ile
Asn Tyr Arg Thr Xaa Xaa Xaa Xaa Xaa Xaa Xaa 210 215
220207217PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 207Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Asp
Ala Gly Ala Val Thr Tyr Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr Val Pro Gly Gly
Val Arg Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr
Thr Ile Thr Val Tyr Ala Val Thr Asp Tyr65 70 75 80Lys Pro His Ala
Asp Gly Pro His Thr Tyr His Glu Tyr Pro Ile Ser 85 90 95Ile Asn Tyr
Arg Thr Glu Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala 100 105 110Pro
Ala Pro Ala Pro Ala Pro Ala Val Ser Asp Val Pro Arg Asp Leu 115 120
125Glu Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Ser Ala
130 135 140Arg Leu Lys Val Ala Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly145 150 155 160Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro
Lys Asn Val Tyr Thr 165 170 175Ala Thr Ile Ser Gly Leu Lys Pro Gly
Val Asp Tyr Thr Ile Thr Val 180 185 190Tyr Ala Val Thr Arg Phe Arg
Asp Tyr Gln Pro Ile Ser Ile Asn Tyr 195 200 205Arg Thr Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 210 215208228PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 208Met Gly Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu
Leu Ile Ser Trp Ser Ala Arg Leu Lys Val Ala Arg 20 25 30Tyr Tyr Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe
Thr Val Pro Lys Asn Val Tyr Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys
Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Arg Phe65 70 75
80Arg Asp Tyr Gln Pro Ile Ser Ile Asn Tyr Arg Thr Glu Ile Asp Lys
85 90 95Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly
Ser 100 105 110Gly Ser Gly Ser Val Ser Asp Val Pro Arg Asp Leu Glu
Val Val Ala 115 120 125Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Asp
Ala Gly Ala Val Thr 130 135 140Tyr Gln Tyr Tyr Arg Ile Thr Tyr Gly
Glu Thr Gly Gly Asn Ser Pro145 150 155 160Val Gln Glu Phe Thr Val
Pro Gly Gly Val Arg Thr Ala Thr Ile Ser 165 170 175Gly Leu Lys Pro
Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr 180 185 190Asp Tyr
Lys Pro His Ala Asp Gly Pro His Thr Tyr His Glu Tyr Pro 195 200
205Ile Ser Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Cys Gln His His
210 215 220His His His His225209222PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
209Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Glu Val Val Ala Ala
Thr1
5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Ser Ala Arg Leu Lys Val Ala
Arg 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln 35 40 45Glu Phe Thr Val Pro Lys Asn Val Tyr Thr Ala Thr Ile
Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala
Val Thr Arg Phe65 70 75 80Arg Asp Tyr Gln Pro Ile Ser Ile Asn Tyr
Arg Thr Glu Ile Asp Lys 85 90 95Gly Ser Gly Ser Gly Ser Gly Ser Gly
Ser Gly Ser Gly Ser Gly Ser 100 105 110Gly Ser Gly Ser Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ala 115 120 125Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Asp Ala Gly Ala Val Thr 130 135 140Tyr Gln Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro145 150 155
160Val Gln Glu Phe Thr Val Pro Gly Gly Val Arg Thr Ala Thr Ile Ser
165 170 175Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala
Val Thr 180 185 190Asp Tyr Lys Pro His Ala Asp Gly Pro His Thr Tyr
His Glu Tyr Pro 195 200 205Ile Ser Ile Asn Tyr Arg Thr Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 210 215 220210217PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 210Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu
Leu Ile Ser Trp Ser Ala Arg Leu Lys Val Ala Arg 20 25 30Tyr Tyr Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe
Thr Val Pro Lys Asn Val Tyr Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys
Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Arg Phe65 70 75
80Arg Asp Tyr Gln Pro Ile Ser Ile Asn Tyr Arg Thr Glu Pro Ala Pro
85 90 95Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala
Val 100 105 110Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr
Pro Thr Ser 115 120 125Leu Leu Ile Ser Trp Asp Ala Gly Ala Val Thr
Tyr Gln Tyr Tyr Arg 130 135 140Ile Thr Tyr Gly Glu Thr Gly Gly Asn
Ser Pro Val Gln Glu Phe Thr145 150 155 160Val Pro Gly Gly Val Arg
Thr Ala Thr Ile Ser Gly Leu Lys Pro Gly 165 170 175Val Asp Tyr Thr
Ile Thr Val Tyr Ala Val Thr Asp Tyr Lys Pro His 180 185 190Ala Asp
Gly Pro His Thr Tyr His Glu Tyr Pro Ile Ser Ile Asn Tyr 195 200
205Arg Thr Xaa Xaa Xaa Xaa Xaa Xaa Xaa 210 215211222PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
211Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1
5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Ser Ala Arg Leu Lys Val Ala
Arg 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln 35 40 45Glu Phe Thr Val Pro Lys Asn Val Tyr Thr Ala Thr Ile
Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala
Val Thr Arg Phe65 70 75 80Arg Asp Tyr Gln Pro Ile Ser Ile Asn Tyr
Arg Thr Glu Ile Glu Lys 85 90 95Gly Ser Gly Cys Gly Ser Gly Ser Gly
Ser Gly Ser Gly Ser Gly Ser 100 105 110Gly Ser Gly Ser Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ala 115 120 125Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Trp Ala Pro Val Asp Arg 130 135 140Tyr Gln Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro145 150 155
160Val Gln Glu Phe Thr Val Pro Arg Asp Val Tyr Thr Ala Thr Ile Ser
165 170 175Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala
Val Thr 180 185 190Asp Tyr Lys Pro His Ala Asp Gly Pro His Thr Tyr
His Glu Ser Pro 195 200 205Ile Ser Ile Asn Tyr Arg Thr Glu His His
His His His His 210 215 220212226PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 212Met Gly Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu
Leu Ile Ser Trp Ser Ala Arg Leu Lys Val Ala Arg 20 25 30Tyr Tyr Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe
Thr Val Pro Lys Asn Val Tyr Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys
Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Arg Phe65 70 75
80Arg Asp Tyr Gln Pro Ile Ser Ile Asn Tyr Arg Thr Glu Ile Glu Lys
85 90 95Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly
Ser 100 105 110Gly Ser Gly Ser Val Ser Asp Val Pro Arg Asp Leu Glu
Val Val Ala 115 120 125Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Trp
Ala Pro Val Asp Arg 130 135 140Tyr Gln Tyr Tyr Arg Ile Thr Tyr Gly
Glu Thr Gly Gly Asn Ser Pro145 150 155 160Val Gln Glu Phe Thr Val
Pro Arg Asp Val Tyr Thr Ala Thr Ile Ser 165 170 175Gly Leu Lys Pro
Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr 180 185 190Asp Tyr
Lys Pro His Ala Asp Gly Pro His Thr Tyr His Glu Ser Pro 195 200
205Ile Ser Ile Asn Tyr Arg Thr Glu Gly Ser Gly Cys His His His His
210 215 220His His225213222PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 213Met Gly Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu
Leu Ile Ser Trp Ser Ala Arg Leu Lys Val Ala Arg 20 25 30Tyr Tyr Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe
Thr Val Pro Lys Asn Val Tyr Thr Ala Thr Ile Cys Gly Leu 50 55 60Lys
Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Arg Phe65 70 75
80Arg Asp Tyr Gln Pro Ile Ser Ile Asn Tyr Arg Thr Glu Ile Glu Lys
85 90 95Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly
Ser 100 105 110Gly Ser Gly Ser Val Ser Asp Val Pro Arg Asp Leu Glu
Val Val Ala 115 120 125Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Trp
Ala Pro Val Asp Arg 130 135 140Tyr Gln Tyr Tyr Arg Ile Thr Tyr Gly
Glu Thr Gly Gly Asn Ser Pro145 150 155 160Val Gln Glu Phe Thr Val
Pro Arg Asp Val Tyr Thr Ala Thr Ile Ser 165 170 175Gly Leu Lys Pro
Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr 180 185 190Asp Tyr
Lys Pro His Ala Asp Gly Pro His Thr Tyr His Glu Ser Pro 195 200
205Ile Ser Ile Asn Tyr Arg Thr Glu His His His His His His 210 215
220214222PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 214Met Gly Val Ser Asp Val Pro Arg Asp Leu
Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Ser
Ala Arg Leu Lys Val Ala Arg 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr Val Pro Lys Asn
Val Tyr Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr
Thr Ile Thr Val Tyr Ala Val Thr Arg Phe65 70 75 80Arg Asp Tyr Gln
Pro Ile Ser Ile Asn Tyr Arg Thr Glu Ile Glu Lys 85 90 95Gly Ser Gly
Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser 100 105 110Gly
Ser Gly Ser Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala 115 120
125Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Trp Ala Pro Val Asp Arg
130 135 140Tyr Gln Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn
Ser Pro145 150 155 160Val Gln Glu Phe Thr Val Pro Arg Asp Val Tyr
Thr Ala Thr Ile Cys 165 170 175Gly Leu Lys Pro Gly Val Asp Tyr Thr
Ile Thr Val Tyr Ala Val Thr 180 185 190Asp Tyr Lys Pro His Ala Asp
Gly Pro His Thr Tyr His Glu Ser Pro 195 200 205Ile Ser Ile Asn Tyr
Arg Thr Glu His His His His His His 210 215 220215222PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
215Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1
5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Ser Ala Arg Leu Lys Val Ala
Arg 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln 35 40 45Glu Phe Thr Val Pro Lys Asn Val Tyr Thr Ala Thr Ile
Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala
Val Thr Arg Phe65 70 75 80Arg Asp Tyr Gln Pro Ile Cys Ile Asn Tyr
Arg Thr Glu Ile Glu Lys 85 90 95Gly Ser Gly Ser Gly Ser Gly Ser Gly
Ser Gly Ser Gly Ser Gly Ser 100 105 110Gly Ser Gly Ser Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ala 115 120 125Ala Thr Pro Thr Ser
Leu Leu Ile Ser Trp Trp Ala Pro Val Asp Arg 130 135 140Tyr Gln Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro145 150 155
160Val Gln Glu Phe Thr Val Pro Arg Asp Val Tyr Thr Ala Thr Ile Ser
165 170 175Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala
Val Thr 180 185 190Asp Tyr Lys Pro His Ala Asp Gly Pro His Thr Tyr
His Glu Ser Pro 195 200 205Ile Ser Ile Asn Tyr Arg Thr Glu His His
His His His His 210 215 220216222PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 216Met Gly Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu
Leu Ile Ser Trp Ser Ala Arg Leu Lys Val Ala Arg 20 25 30Tyr Tyr Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe
Thr Val Pro Lys Asn Val Tyr Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys
Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Arg Phe65 70 75
80Arg Asp Tyr Gln Pro Ile Ser Ile Asn Tyr Arg Thr Glu Ile Glu Lys
85 90 95Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly
Ser 100 105 110Gly Ser Gly Ser Val Ser Asp Val Pro Arg Asp Leu Glu
Val Val Ala 115 120 125Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Trp
Ala Pro Val Asp Arg 130 135 140Tyr Gln Tyr Tyr Arg Ile Thr Tyr Gly
Glu Thr Gly Gly Asn Ser Pro145 150 155 160Val Gln Glu Phe Thr Val
Pro Arg Asp Val Tyr Thr Ala Thr Ile Ser 165 170 175Gly Leu Lys Pro
Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr 180 185 190Asp Tyr
Lys Pro His Ala Asp Gly Pro His Thr Tyr His Glu Ser Pro 195 200
205Ile Cys Ile Asn Tyr Arg Thr Glu His His His His His His 210 215
2202174PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 217Glu Ile Glu Lys121820PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 218Gly
Ser Gly Cys Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser1 5 10
15Gly Ser Gly Ser 20219108PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 219Met Gly Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu
Leu Ile Ser Trp Gln Val Pro Arg Pro Met Tyr Gln 20 25 30Tyr Tyr Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe
Thr Val Pro Tyr Asp Val Tyr Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys
Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Asp Ser65 70 75
80Tyr Asn Pro Ala Thr His Glu Tyr Lys Tyr His Gln Thr Pro Ile Ser
85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln 100
105220108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 220Met Gly Val Ser Asp Val Pro Arg Asp Leu
Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Gln
Val Pro Arg Pro Met Tyr Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr Val Pro Tyr Asp
Val His Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr
Thr Ile Thr Val Tyr Ala Val Thr Asp Ser65 70 75 80Tyr Asn Pro Ala
Thr His Glu Tyr Lys Tyr His Gln Thr Pro Ile Ser 85 90 95Ile Asn Tyr
Arg Thr Glu Ile Asp Lys Pro Ser Gln 100 105221108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
221Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1
5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Gln Val Pro Arg Pro Met Tyr
Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln 35 40 45Glu Phe Thr Val Pro Tyr Asp Leu Thr Thr Ala Thr Ile
Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala
Val Thr Asp Ser65 70 75 80Tyr Asn Pro Ala Thr His Glu Tyr Lys Tyr
His Gln Thr Pro Ile Ser 85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys
Pro Ser Gln 100 105222108PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 222Met Gly Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu
Leu Ile Ser Trp Glu Ala Asn Pro Ser Arg Tyr Gln 20 25 30Tyr Tyr Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe
Thr Val Pro His Asp Leu Asn Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys
Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Asp Ser65 70 75
80Tyr Asn Pro Ala Thr His Glu Tyr Lys Tyr His Gln Thr Pro Ile Ser
85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln 100
105223108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 223Met Gly Val Ser Asp Val Pro Arg Asp Leu
Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Tyr
Pro Gly Ser Arg Thr Tyr Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr Val Pro Gly Gly
Val Arg Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr
Thr Ile Thr Val Tyr Ala Val Thr Asp Tyr65 70 75 80Tyr His Pro Ala
Thr Tyr Glu His Glu Tyr His
Ala His Pro Ile Ser 85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro
Ser Gln 100 105224108PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 224Met Gly Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu
Leu Ile Ser Trp Thr Pro Ala Asn Lys Ser Tyr Gln 20 25 30Tyr Tyr Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe
Thr Val Pro Tyr Asp Gly Thr Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys
Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Asp His65 70 75
80Lys Pro His Ala Asp Gly Pro His Thr Tyr His Glu Tyr Pro Ile Ser
85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln 100
105225108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 225Met Gly Val Ser Asp Val Pro Arg Asp Leu
Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Gln
Val Pro Arg Pro Met Tyr Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr Val Pro His Asp
Val Tyr Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr
Thr Ile Thr Val Tyr Ala Val Thr Asp Ser65 70 75 80Tyr Asn Pro Ala
Thr His Glu Tyr Lys Tyr His Gln Thr Pro Ile Ser 85 90 95Ile Asn Tyr
Arg Thr Glu Ile Asp Lys Pro Ser Gln 100 105226108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
226Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1
5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Gln Val Pro Arg Pro Met Tyr
Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln 35 40 45Glu Phe Thr Val Pro Gly Gln Val Tyr Thr Ala Thr Ile
Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala
Val Thr Asp Ser65 70 75 80Tyr Asn Pro Ala Thr His Glu Tyr Lys Tyr
His Gln Thr Pro Ile Ser 85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys
Pro Ser Gln 100 105227108PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 227Met Gly Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu
Leu Ile Ser Trp Gln Val Pro Arg Pro Met Tyr Gln 20 25 30Tyr Tyr Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe
Thr Val Pro Tyr Asp Val Thr Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys
Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Asp Ser65 70 75
80Tyr Asn Pro Ala Thr His Glu Tyr Lys Tyr His Gln Thr Pro Ile Ser
85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln 100
105228108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 228Met Gly Val Ser Asp Val Pro Arg Asp Leu
Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Gln
Val Pro Arg Pro Met Tyr Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr Val Pro His Asp
Leu Arg Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr
Thr Ile Thr Val Tyr Ala Val Thr Asp Ser65 70 75 80Tyr Asn Pro Ala
Thr His Glu Tyr Lys Tyr His Gln Thr Pro Ile Ser 85 90 95Ile Asn Tyr
Arg Thr Glu Ile Asp Lys Pro Ser Gln 100 105229108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
229Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1
5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Gln Val Pro Arg Pro Met Tyr
Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln 35 40 45Glu Phe Thr Val Pro Tyr Asp Leu Val Thr Ala Thr Ile
Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala
Val Thr Asp His65 70 75 80Lys Pro His Ala Asp Gly Pro His Thr Tyr
His Glu Ser Pro Ile Ser 85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys
Pro Ser Gln 100 105230104PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 230Met Gly Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu
Leu Ile Ser Trp Leu Pro Gly Lys Leu Arg Tyr Gln 20 25 30Tyr Tyr Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe
Thr Val Pro His Asp Leu Arg Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys
Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Asn Met65 70 75
80Met His Val Glu Tyr Ser Glu Tyr Pro Ile Ser Ile Asn Tyr Arg Thr
85 90 95Glu Ile Asp Lys Pro Ser Gln His 100231103PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
231Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1
5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Val Ala Gly Ala Glu Asp Tyr
Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln 35 40 45Glu Phe Thr Val Pro His Asp Leu Val Thr Ala Thr Ile
Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala
Val Thr Asp Met65 70 75 80Met His Val Glu Tyr Thr Glu His Pro Ile
Ser Ile Asn Tyr Arg Thr 85 90 95Glu Ile Asp Lys Pro Ser Gln
100232108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 232Met Gly Val Ser Asp Val Pro Arg Asp Leu
Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Gln
Val Pro Arg Pro Met Tyr Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr Val Pro Gly Met
Val His Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr
Thr Ile Thr Val Tyr Ala Val Thr Asp His65 70 75 80Lys Pro His Ala
Asp Gly Pro His Thr Tyr His Glu Ser Pro Ile Ser 85 90 95Ile Asn Tyr
Arg Thr Glu Ile Asp Lys Pro Ser Gln 100 105233108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
233Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1
5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Phe Val Thr His Val Ala Tyr
Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln 35 40 45Glu Phe Thr Val Pro Gly Gly Leu Ser Thr Ala Thr Ile
Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala
Val Thr Asp Tyr65 70 75 80Lys Pro His Ala Asp Gly Pro His Thr Tyr
His Glu Ser Pro Ile Ser 85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys
Pro Ser Gln 100 105234108PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 234Met Gly Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu
Leu Ile Ser Trp Glu Thr Glu Ser Asn Ala Tyr Gln 20 25 30Tyr Tyr Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe
Thr Val Pro Gly Gln Ile Tyr Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys
Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Asp Tyr65 70 75
80Lys Pro His Ala Asp Gly Pro His Thr Tyr His Glu Ser Pro Ile Ser
85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln 100
105235108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 235Met Gly Val Ser Asp Val Pro Arg Asp Leu
Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Met
Thr Ser Pro Ser Val Tyr Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr Val Pro Gly Pro
Val Gln Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr
Thr Ile Thr Val Tyr Ala Val Thr Asp Tyr65 70 75 80Lys Glu His Gln
His Ala Pro His Gln Tyr Thr Ala His Pro Ile Ser 85 90 95Ile Asn Tyr
Arg Thr Glu Ile Asp Lys Pro Ser Gln 100 105236108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
236Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1
5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Asp Ser Gly Arg Gly Ser Tyr
Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln 35 40 45Glu Phe Thr Val Pro Gly Pro Val His Thr Ala Thr Ile
Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala
Val Thr Asp His65 70 75 80Lys Pro His Ala Asp Gly Pro His Thr Tyr
His Glu Ser Pro Ile Ser 85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys
Pro Ser Gln 100 105237108PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 237Met Gly Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu
Leu Ile Ser Trp Ser Thr Gly Arg Thr Thr Tyr Gln 20 25 30Tyr Tyr Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe
Thr Val Pro His Asp Leu Asp Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys
Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Asp Ser65 70 75
80Tyr Asn Pro Ala Thr His Glu Tyr Lys Tyr His Gln Thr Pro Ile Ser
85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln 100
105238103PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 238Met Gly Val Ser Asp Val Pro Arg Asp Leu
Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Gln
Val Pro Arg Pro Met Tyr Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr Val Pro Tyr Asp
Val Ile Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr
Thr Ile Thr Val Tyr Ala Val Thr Asp Met65 70 75 80Met His Val Glu
Tyr Ala Glu Tyr Pro Ile Ser Ile Asn Tyr Arg Thr 85 90 95Glu Ile Asp
Lys Pro Ser Gln 100239108PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 239Met Gly Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu
Leu Ile Ser Trp Gln Val Pro Arg Pro Met Tyr Gln 20 25 30Tyr Tyr Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe
Thr Val Pro Gly Gln Val Pro Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys
Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Asp Ser65 70 75
80Tyr Asn Pro Ala Thr His Glu Tyr Lys Tyr His Gln Thr Pro Ile Ser
85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln 100
105240103PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 240Met Gly Val Ser Asp Val Pro Arg Asp Leu
Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Gly
Tyr Gln Ser Gly Gly Tyr Thr 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr Val Pro His Asp
Leu Arg Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr
Thr Ile Thr Val Tyr Ala Val Thr Asp Tyr65 70 75 80Ala Tyr Lys Glu
Tyr Gln Glu His Pro Ile Ser Ile Asn Tyr Arg Thr 85 90 95Glu Ile Asp
Lys Pro Ser Gln 100241103PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 241Met Gly Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu
Leu Ile Ser Trp Trp Ile Gly Ile Pro Val Tyr Gln 20 25 30Tyr Tyr Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe
Thr Val Pro Tyr Asp Gly Lys Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys
Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Asp Met65 70 75
80Met His Val Glu Tyr Ala Glu Tyr Pro Ile Ser Ile Asn Tyr Arg Thr
85 90 95Glu Ile Asp Lys Pro Ser Gln 100242108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
242Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1
5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Ser Lys Gly Ser Lys Ser Tyr
Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln 35 40 45Glu Phe Thr Val Pro Tyr His Val Tyr Thr Ala Thr Ile
Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala
Val Thr Asp Tyr65 70 75 80Tyr Asn Pro Ala Thr Tyr Glu Tyr Ile Tyr
Leu Thr Thr Pro Ile Ser 85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys
Pro Ser Gln 100 105243108PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 243Met Gly Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu
Leu Ile Ser Trp Asn Pro Gly Ser Lys Ser Tyr Gln 20 25 30Tyr Tyr Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe
Thr Val Pro Gly Gly Val Arg Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys
Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Asp Phe65 70 75
80Tyr Asn Pro Asp Thr His Glu Tyr Leu Tyr Asn Gln Tyr Pro Ile Ser
85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln 100
105244108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 244Met Gly Val Ser Asp Val Pro Arg Asp Leu
Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Gln
Pro Gly Thr Thr His Tyr Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr Val Pro Tyr Asp
Leu Met Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr
Thr Ile Thr Val Tyr Ala Val Thr Asp Tyr65 70 75
80Tyr Asn Pro Asn Thr Tyr Glu Tyr Ile Tyr Leu Thr Thr Pro Ile Ser
85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln 100
105245108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 245Met Gly Val Ser Asp Val Pro Arg Asp Leu
Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Ala
Ile Gly Thr Ile Val Tyr Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr Val Pro Ala Gly
Val Tyr Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr
Thr Ile Thr Val Tyr Ala Val Thr Asp Tyr65 70 75 80Tyr Asp Trp Ala
Thr His Glu Tyr Asn Tyr His Thr Ala Pro Ile Ser 85 90 95Ile Asn Tyr
Arg Thr Glu Ile Asp Lys Pro Ser Gln 100 105246108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
246Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1
5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Thr Tyr Asn Asp Gly Ser Tyr
Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln 35 40 45Glu Phe Thr Val Pro Tyr Ala Val Tyr Thr Ala Thr Ile
Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala
Val Thr Asp Phe65 70 75 80Tyr Asn Pro Ala Thr Tyr Glu Tyr Ile Tyr
His Thr Thr Pro Ile Ser 85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys
Pro Ser Gln 100 105247108PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 247Met Gly Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu
Leu Ile Ser Trp Val Ser Leu Val Gly Phe Tyr Gln 20 25 30Tyr Tyr Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe
Thr Val Pro Gly Gly Val His Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys
Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Asp Tyr65 70 75
80Lys Pro His Ala Asp Gly Pro His Thr Tyr His Glu Ser Pro Ile Ser
85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln 100
105248103PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 248Met Gly Val Ser Asp Val Pro Arg Asp Leu
Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Ala
Ser Arg Lys Glu Val Tyr Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr Val Pro Gly Trp
Leu Asn Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr
Thr Ile Thr Val Tyr Ala Val Thr Asp Tyr65 70 75 80Met His Val Glu
Tyr Ala Glu Tyr Pro Ile Ser Ile Asn Tyr Arg Thr 85 90 95Glu Ile Asp
Lys Pro Ser Gln 100249108PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 249Met Gly Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu
Leu Ile Ser Trp Leu Ala Pro Phe Trp Arg Tyr Gln 20 25 30Tyr Tyr Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe
Thr Val Pro Gly Gly Val Arg Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys
Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Asp Tyr65 70 75
80Lys Pro His Ala Asp Gly Pro His Thr Tyr His Glu Ser Pro Ile Ser
85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln 100
105250108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 250Met Gly Val Ser Asp Val Pro Arg Asp Leu
Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Thr
Pro Pro Gly His Gln His Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr Val Pro Gly Gln
Val Thr Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr
Thr Ile Thr Val Tyr Ala Val Thr Asp Tyr65 70 75 80Tyr Asn Pro Ala
Thr His Tyr Tyr Thr Tyr Tyr Thr Thr Pro Ile Ser 85 90 95Ile Asn Tyr
Arg Thr Glu Ile Asp Lys Pro Ser Gln 100 105251108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
251Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1
5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Glu Ser Gly Ser Arg Thr Tyr
Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln 35 40 45Glu Phe Thr Val Pro Gly Gly Val His Thr Ala Thr Ile
Ser Gly Leu 50 55 60Lys Thr Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala
Val Thr Asp Tyr65 70 75 80Lys Pro His Ala Asp Gly Pro His Thr Tyr
His Glu Tyr Pro Ile Ser 85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys
Pro Ser Gln 100 105252108PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 252Met Gly Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu
Leu Ile Ser Trp Glu Arg Thr Ser Thr His Tyr Gln 20 25 30Tyr Tyr Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe
Thr Val Pro Gly Arg Val Tyr Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys
Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Asp Tyr65 70 75
80Tyr Asn Pro Ala Thr His Glu Tyr Lys Tyr His Gln Thr Pro Ile Ser
85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln 100
105253108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 253Met Gly Val Ser Asp Val Pro Arg Asp Leu
Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Asn
Ala Arg Thr Asp Ala Tyr Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr Val Pro Arg Asp
Leu Glu Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr
Thr Ile Thr Val Tyr Ala Val Thr Asp Tyr65 70 75 80Lys Pro His Ala
Asp Gly Pro His Thr Tyr Gln Glu Ser Pro Ile Ser 85 90 95Ile Asn Tyr
Arg Thr Glu Ile Asp Lys Pro Ser Gln 100 105254108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
254Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1
5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Gln Val Ser Ala Phe Arg Tyr
Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln 35 40 45Glu Phe Thr Val Pro Gly Met Val Ser Thr Ala Thr Ile
Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala
Val Thr Asp Tyr65 70 75 80Lys Pro His Ala Asp Gly Pro His Thr Tyr
Ser Glu Tyr Pro Ile Ser 85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys
Pro Ser Gln 100 105255108PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 255Met Gly Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu
Leu Ile Ser Trp Val Leu Gly Arg Arg Val Tyr Gln 20 25 30Tyr Tyr Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe
Thr Val Pro Gly Ala Val Tyr Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys
Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Asp Tyr65 70 75
80Phe Asn Pro Ala Thr His Glu Tyr Gln Tyr Glu Leu Thr Pro Ile Ser
85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln 100
105256108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 256Met Gly Val Ser Asp Val Pro Arg Asp Leu
Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Thr
Pro Pro Asn Ser Gly His Asn 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr Val Pro His Asp
Leu Thr Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr
Thr Ile Thr Val Tyr Ala Val Thr Asp Tyr65 70 75 80Tyr Asn Pro Asn
Thr Tyr Glu Tyr Thr Tyr Gln Phe Thr Pro Ile Ser 85 90 95Ile Asn Tyr
Arg Thr Glu Ile Asp Lys Pro Ser Gln 100 105257108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
257Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1
5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Val Val Pro Asn Trp Met Tyr
Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln 35 40 45Glu Phe Thr Val Pro Gly Met Leu Glu Thr Ala Thr Ile
Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala
Val Thr Asp Tyr65 70 75 80Tyr Asn Pro Thr Thr Tyr Glu Tyr Thr Tyr
Phe Thr Tyr Pro Ile Ser 85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys
Pro Ser Gln 100 105258108PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 258Met Gly Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu
Leu Ile Ser Trp Ser Gly Gly Phe Met Arg Tyr Gln 20 25 30Tyr Tyr Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe
Thr Val Pro Gly Gln Val Tyr Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys
Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Asp Tyr65 70 75
80Lys Pro His Ala Asp Gly Pro His Thr Tyr His Glu Ser Pro Ile Ser
85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln 100
105259108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 259Met Gly Val Ser Asp Val Pro Arg Asp Leu
Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Asp
Ser Glu Gly Pro Ser Tyr Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr Val Pro Tyr Ala
Val Tyr Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr
Thr Ile Thr Val Tyr Ala Val Thr Asp Tyr65 70 75 80Tyr Asn Pro Arg
Thr His Glu Leu Phe Phe Gln Gln Tyr Pro Ile Ser 85 90 95Ile Asn Tyr
Arg Thr Glu Ile Asp Lys Pro Ser Gln 100 105260108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
260Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1
5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Gln Val Pro Arg Pro Met Tyr
Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu Lys Gly Gly Asn Ser Pro
Val Gln 35 40 45Glu Phe Thr Val Pro His Asp Leu Arg Thr Ala Thr Ile
Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala
Val Thr Asp Tyr65 70 75 80Tyr Asp Pro Thr Ser Asn Leu Tyr Asn Tyr
Asn Gln Thr Pro Ile Ser 85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys
Pro Ser Gln 100 105261108PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 261Met Gly Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu
Leu Ile Ser Trp Gln Val Gly Ser Val Val Tyr Gln 20 25 30Tyr Tyr Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe
Thr Val Pro Arg Asp Val Leu Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys
Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Asp Tyr65 70 75
80Lys Pro Lys Pro Asp Gly Pro His Ile Tyr Gln Ala Val Pro Ile Ser
85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln 100
105262108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 262Met Gly Val Ser Asp Val Pro Arg Asp Leu
Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Asn
Pro Ala Ser Lys Asp Tyr Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr Val Pro Gly Gln
Val Pro Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr
Thr Ile Thr Val Tyr Ala Val Thr Asp Phe65 70 75 80Tyr Asn Pro Ala
Thr His Glu Tyr Lys Tyr Asp Ser Thr Pro Ile Ser 85 90 95Ile Asn Tyr
Arg Thr Glu Ile Asp Lys Pro Ser Gln 100 105263108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
263Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1
5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Arg Ser Ser Ala Thr Ala Tyr
Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln 35 40 45Glu Phe Thr Val Pro Gly Arg Val Tyr Thr Ala Thr Ile
Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala
Val Thr Asp Phe65 70 75 80Phe Asn Trp Ala Thr His Glu Tyr Ile Tyr
His Ser Thr Pro Ile Ser 85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys
Pro Ser Gln 100 105264108PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 264Met Gly Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu
Leu Ile Ser Trp His Ser Gly Pro Arg Glu Tyr Gln 20 25 30Tyr Tyr Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe
Thr Val Pro Gly Gln Val Tyr Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys
Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Asp Phe65 70 75
80Phe Asn Pro Ile Thr His Tyr Tyr Tyr Tyr Glu Leu Thr Pro Ile Ser
85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln 100
105265108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 265Met Gly Val Ser Asp Val Pro Arg Asp Leu
Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Thr
Val Gly Leu Ser Val Tyr Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr Val Pro Gly Met
Val Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro Ser Val Asp
Tyr
Thr Ile Thr Val Tyr Ala Val Thr Asp Tyr65 70 75 80Lys Pro His Ala
Asp Gly Pro His Thr Tyr His Glu Tyr Pro Ile Ser 85 90 95Ile Asn Tyr
Arg Thr Glu Ile Asp Lys Pro Ser Gln 100 105266108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
266Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1
5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Gly Gly His Arg Ala Val Tyr
Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln 35 40 45Glu Phe Thr Val Pro Gly Ala Val Tyr Thr Ala Thr Ile
Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala
Val Thr Asp Tyr65 70 75 80Tyr Asn Pro Asp Thr His Glu Tyr Lys Tyr
His Gln Tyr Pro Ile Ser 85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys
Pro Ser Gln 100 105267103PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 267Met Gly Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ser Ala Thr1 5 10 15Pro Thr Ser Leu
Leu Ile Ser Trp Gln Val Pro Arg Pro Met Tyr Gln 20 25 30Tyr Tyr Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe
Thr Val Pro Gly Gly Val Arg Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys
Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Asp Tyr65 70 75
80Trp Phe Lys Glu Tyr Arg Glu Asp Pro Ile Ser Ile Asn Tyr Arg Thr
85 90 95Glu Ile Asp Lys Pro Ser Gln 100268108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
268Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1
5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Ser Val Gly Gly Met Ile Tyr
Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln 35 40 45Glu Phe Thr Val Pro Gly Met Val Thr Thr Ala Thr Ile
Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala
Val Thr Asp Tyr65 70 75 80Tyr Asn Pro Ala Thr His Glu Tyr Lys Tyr
His Gln Thr Pro Ile Ser 85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys
Pro Ser Gln 100 105269108PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 269Met Gly Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu
Leu Ile Ser Trp Lys Ala Ser Tyr Thr Gly Tyr Asn 20 25 30Tyr Tyr Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe
Thr Val Pro Arg Asp Val Met Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys
Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Asp Phe65 70 75
80Tyr Asn Pro Asp Thr His Gln Tyr Thr Tyr Arg Arg Ile Pro Ile Ser
85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln 100
105270108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 270Met Gly Val Ser Asp Val Pro Arg Asp Leu
Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Ser
Val Gly Gln Val Phe Tyr Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr Val Pro Tyr Asp
Val Tyr Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr
Thr Ile Thr Val Tyr Ala Val Thr Asp Tyr65 70 75 80Tyr Asn Pro Ala
Thr His Glu Tyr Lys Tyr His Gln Thr Pro Ile Ser 85 90 95Ile Asn Tyr
Arg Thr Glu Ile Asp Lys Pro Ser Gln 100 105271108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
271Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1
5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Tyr Ser Gly Asp Tyr His Tyr
Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln 35 40 45Glu Phe Thr Val Pro His Asp Leu Glu Thr Ala Thr Ile
Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala
Val Thr Asp Tyr65 70 75 80Tyr Asn Pro Ala Thr His Tyr Tyr Lys Tyr
Glu Gln Thr Pro Ile Ser 85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys
Pro Ser Gln 100 105272108PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 272Met Gly Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu
Leu Ile Ser Trp Ile Val Gln Gly Gly Arg Tyr Gln 20 25 30Tyr Tyr Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe
Thr Val Pro Gly Met Val Thr Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys
Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Asp Tyr65 70 75
80Tyr Asn Pro Ser Thr His Glu Tyr Lys Tyr His Gln Thr Pro Ile Ser
85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln 100
105273108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 273Met Gly Val Ser Asp Val Pro Arg Asp Leu
Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Ser
Ala Val Arg Trp Arg Tyr Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr Val Pro Gly Gly
Val Arg Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr
Thr Ile Thr Val Tyr Ala Val Thr Asp Phe65 70 75 80Tyr Asn Pro Arg
Thr His Val Tyr Ile Tyr Asp Gln Phe Pro Ile Ser 85 90 95Ile Asn Tyr
Arg Thr Glu Ile Asp Lys Pro Ser Gln 100 105274108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
274Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1
5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Arg Ala Arg Arg Leu Gln Tyr
Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln 35 40 45Glu Phe Thr Val Pro Gly Met Val Thr Thr Ala Thr Ile
Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala
Val Thr Asp Phe65 70 75 80Tyr Asn Pro Ala Thr Met Glu Tyr Thr Tyr
Gln Arg Thr Pro Ile Ser 85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys
Pro Ser Gln 100 105275108PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 275Met Gly Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu
Leu Ile Ser Trp Leu Gln Pro Leu Trp Arg Tyr Gln 20 25 30Tyr Tyr Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe
Thr Val Pro Gly Gly Leu Asp Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys
Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Asp Tyr65 70 75
80Lys Pro His Val Asp Gly Pro His Ala Tyr His Glu Tyr Pro Ile Ser
85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln 100
105276108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 276Met Gly Val Ser Asp Val Pro Arg Asp Leu
Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Asp
Ala Ser Gln Gly Asn Tyr Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr Val Pro Gly Ala
Val Lys Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr
Thr Ile Thr Val Tyr Ala Val Thr Asp Phe65 70 75 80Phe Asn Pro Ala
Thr His Glu Tyr Ile Tyr His Thr Thr Pro Ile Ser 85 90 95Ile Asn Tyr
Arg Thr Glu Ile Asp Lys Pro Ser Gln 100 105277108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
277Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1
5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Cys Leu Asp Gly Gln Leu Tyr
Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln 35 40 45Glu Phe Thr Val Pro Gly Ser Ile Val Thr Ala Thr Ile
Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala
Val Thr Asp Trp65 70 75 80Tyr Asn Leu Ala Thr His Glu Tyr Asn Tyr
Arg Val Thr Pro Ile Ser 85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys
Pro Ser Gln 100 105278108PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 278Met Gly Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu
Leu Ile Ser Trp Asp Thr Ser Gly Ala Ser Tyr Gln 20 25 30Tyr Tyr Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe
Thr Val Pro Tyr Ser Val Tyr Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys
Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Asp Tyr65 70 75
80Tyr Asp Pro Asp Ser His Tyr Tyr Asn Tyr Asn Met Val Pro Ile Ser
85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln 100
105279108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 279Met Gly Val Ser Asp Val Pro Arg Asp Leu
Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Asp
Ser Gly Asn Gly Thr Tyr Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr Val Pro Tyr Arg
Val Tyr Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr
Thr Ile Thr Val Tyr Ala Val Thr Asp Tyr65 70 75 80Tyr Asn Pro Ala
Thr His Glu Tyr Thr Tyr Glu Leu Arg Pro Ile Ser 85 90 95Ile Asn Tyr
Arg Thr Glu Ile Asp Lys Pro Ser Gln 100 105280108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
280Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Thr Thr1
5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Arg Pro Thr Ser Gln Val Tyr
Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln 35 40 45Glu Phe Thr Val Pro Tyr Asn Val Tyr Thr Ala Thr Ile
Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala
Val Thr Asp Phe65 70 75 80Phe Asn Tyr Ala Thr His Glu Tyr Ile Tyr
His Thr Ile Pro Ile Ser 85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys
Pro Ser Gln 100 105281108PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 281Met Gly Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu
Leu Ile Ser Trp Lys Ser Tyr Gly Ser Ala Tyr Gln 20 25 30Tyr Tyr Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe
Thr Val Pro Gly Asp Leu Gln Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys
Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Ile Thr Asp Tyr65 70 75
80Tyr Asn Pro Asp Thr His Glu Tyr Lys Tyr His Val Ser Pro Ile Ser
85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln 100
105282108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 282Met Gly Val Ser Asp Val Pro Arg Asp Leu
Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Ser
Ser Val Met Gly Leu Tyr Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr Val Pro Tyr Asp
Val Tyr Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr
Thr Ile Thr Val Tyr Ala Val Thr Asp Tyr65 70 75 80Tyr Asn Pro Ser
Thr Tyr Glu Tyr Lys Tyr Asn Thr Thr Pro Ile Ser 85 90 95Ile Asn Tyr
Arg Thr Glu Ile Asp Lys Pro Ser Gln 100 105283108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
283Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1
5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Lys Thr Glu Pro Gly Arg His
Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln 35 40 45Glu Phe Thr Val Pro Gly Gly Val Arg Thr Ala Thr Ile
Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala
Val Thr Asp Trp65 70 75 80Tyr Asn Leu Val Ser His Glu Tyr Val Tyr
His Thr Thr Pro Ile Ser 85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys
Pro Ser Gln 100 105284108PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 284Met Gly Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu
Leu Ile Ser Trp His Ala Gly Met Ala Val Tyr Gln 20 25 30Tyr Tyr Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe
Thr Val Pro Gly Asp Val Leu Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys
Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Asp Phe65 70 75
80Phe Asn Pro Val Thr His Glu Tyr Met Tyr His Thr Ile Pro Ile Ser
85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln 100
105285108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 285Met Gly Val Ser Asp Val Pro Arg Asp Leu
Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Val
Ser Ala Arg Gly Arg Tyr Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr Val Pro Gly Gly
Val Arg Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr
Thr Ile Thr Val Tyr Ala Val Thr Asp Tyr65 70 75 80Tyr Asn Leu Glu
Thr Tyr Glu Tyr His Tyr Tyr Arg Thr Pro Ile Ser 85 90 95Ile Asn Tyr
Arg Thr Glu Ile Asp Lys Pro Ser Gln 100 105286108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
286Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1
5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Trp Phe Gly Thr Ser Ser Tyr
Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln 35 40 45Glu Phe Thr Val Pro Gly Asp Leu Lys Thr Ala Thr Ile
Ser Gly
Leu 50 55 60Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr
Asp Tyr65 70 75 80Phe Asn Pro Val Thr His Glu Tyr Glu Tyr His Thr
Thr Pro Ile Ser 85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser
Gln 100 105287108PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 287Met Gly Val Ser Asp Val Pro Arg
Asp Leu Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu Leu Ile Ser
Trp Ser Ala Thr Arg Thr Leu Tyr Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr
Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr Val Pro
Tyr Asp Val His Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro Gly Val
Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Asp Tyr65 70 75 80Tyr Asn
Met Val Thr Tyr Glu Tyr Asn Tyr His Leu Thr Pro Ile Ser 85 90 95Ile
Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln 100
105288108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 288Met Gly Val Ser Asp Val Pro Arg Asp Leu
Glu Val Val Val Ala Thr1 5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Thr
Lys Leu Leu Gly Gly Tyr Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr Val Pro Gly Pro
Val Tyr Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr
Thr Ile Thr Val Tyr Ala Val Thr Asp Phe65 70 75 80Phe Asn Pro Arg
Thr His Glu Tyr Gln Tyr His Thr Thr Pro Ile Ser 85 90 95Ile Asn Tyr
Arg Thr Glu Ile Asp Lys Pro Ser Gln 100 105289108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
289Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1
5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Arg Ala Ser Gly Gly Leu Tyr
Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln 35 40 45Glu Phe Thr Val Pro Gly Ser Val Asn Thr Ala Thr Ile
Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala
Val Thr Asp Phe65 70 75 80Tyr Asn Pro Ala Thr Tyr Glu Tyr Ile Tyr
His Thr Thr Pro Ile Ser 85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys
Pro Ser Gln 100 105290108PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 290Met Gly Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu
Leu Ile Ser Trp Ala Ala Gly Arg Ala Thr Tyr Gln 20 25 30Tyr Tyr Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe
Thr Val Pro Tyr Asp Val Thr Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys
Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Asp Phe65 70 75
80Tyr Asn Pro Ala Thr His Glu Tyr Tyr Tyr Glu Thr Thr Pro Ile Ser
85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln 100
105291108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 291Met Gly Val Ser Asp Val Pro Arg Asp Leu
Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Tyr
Ser Gln Pro Leu Thr Tyr Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr Val Pro His Asp
Val Asn Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr
Thr Ile Thr Val Tyr Ala Val Thr Asp Phe65 70 75 80Tyr Asn Pro Glu
Thr His Glu Tyr Thr Tyr His Leu Thr Pro Ile Ser 85 90 95Ile Asn Tyr
Arg Thr Glu Ile Asp Lys Pro Ser Gln 100 105292108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
292Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1
5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Ser Ser Ala Thr Arg Pro Tyr
Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln 35 40 45Glu Phe Thr Val Pro Gly Gly Val Arg Thr Ala Thr Ile
Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala
Val Thr Asp Phe65 70 75 80Phe Asn Pro Thr Thr His Glu Tyr Tyr Tyr
His Thr Thr Pro Ile Ser 85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys
Pro Ser Gln 100 105293108PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 293Met Gly Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu
Leu Ile Ser Trp Ser Val Glu Arg Ser Val Tyr Gln 20 25 30Tyr Tyr Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe
Thr Val Pro Gly Gly Val Arg Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys
Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Asp Tyr65 70 75
80Tyr Asn Pro Ser Thr His Glu Tyr Asn Tyr Leu Thr Thr Pro Ile Ser
85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln 100
105294108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 294Met Gly Val Ser Asp Val Pro Arg Asp Leu
Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Gln
Asp Thr Ser Ser Tyr His Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr Val Pro Gly Gly
Val Arg Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr
Thr Ile Thr Val Tyr Ala Val Thr Asp Tyr65 70 75 80Phe Asn Pro Ser
Thr His Glu Tyr Ile Tyr Arg Thr Ile Pro Ile Ser 85 90 95Ile Asn Tyr
Arg Thr Glu Ile Asp Lys Pro Ser Gln 100 105295108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
295Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1
5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Ser Ser Ser His Arg Arg Tyr
Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln 35 40 45Glu Phe Thr Val Pro Gly Ser Val Ala Thr Ala Thr Ile
Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala
Val Thr Asp Tyr65 70 75 80Phe Asn Pro Asp Thr His Glu Tyr Leu Tyr
His Ala Thr Pro Ile Ser 85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys
Pro Ser Gln 100 105296108PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 296Met Gly Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu
Leu Ile Ser Trp Asp Asn Asn Ser Asn Ser Tyr Gln 20 25 30Tyr Tyr Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe
Thr Val Pro Tyr Asp Leu Arg Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys
Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Asp Tyr65 70 75
80Lys Pro His Thr Glu Gly Glu His Thr Tyr His Glu Ser Pro Ile Ser
85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln 100
105297108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 297Met Gly Val Ser Asp Val Pro Arg Asp Leu
Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Arg
Val Leu Val Asp Met Tyr Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr Val Pro Gly Gly
Val Leu Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr
Thr Ile Thr Val Tyr Ala Val Thr Asp Tyr65 70 75 80Lys Pro His Val
Asp Gly Pro His Thr Tyr Tyr Glu Ser Pro Ile Ser 85 90 95Ile Asn Tyr
Arg Thr Glu Ile Asp Lys Pro Ser Gln 100 105298108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
298Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1
5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Met Phe Val Gly Met Ser Tyr
Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln 35 40 45Glu Phe Thr Val Pro Tyr Gly Val His Thr Ala Thr Ile
Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala
Val Thr Asp Tyr65 70 75 80Phe Asn Pro Ala Thr His Glu Tyr Ile Tyr
His Val Thr Pro Ile Ser 85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys
Pro Ser Gln 100 105299108PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 299Met Gly Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu
Leu Ile Ser Trp Thr Leu His Arg Lys Asn Tyr Gln 20 25 30Tyr Tyr Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe
Thr Val Pro Gly Gly Val Val Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys
Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Asp Tyr65 70 75
80Tyr Asn Pro Ala Thr His Glu Tyr Asp Tyr Arg Thr Thr Pro Ile Ser
85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln 100
105300108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 300Met Gly Val Ser Asp Val Pro Arg Asp Leu
Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Thr
Gln Gly Ser Thr His Tyr Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr Val Pro Gly Met
Val Tyr Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr
Thr Ile Thr Val Tyr Ala Val Thr Asp Tyr65 70 75 80Phe Asp Arg Ser
Thr His Glu Tyr Lys Tyr Arg Thr Thr Pro Ile Ser 85 90 95Ile Asn Tyr
Arg Thr Glu Ile Asp Lys Pro Ser Gln 100 105301108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
301Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1
5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Asp Ser Gly Glu Asn Asn Tyr
Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln 35 40 45Glu Phe Thr Val Pro Gly Gly Val Arg Thr Ala Thr Ile
Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala
Val Thr Asp Tyr65 70 75 80Tyr Asn Pro Lys Thr His Glu Tyr Asn Tyr
Leu Thr Ile Pro Ile Ser 85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys
Pro Ser Gln 100 105302108PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 302Met Gly Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu
Leu Ile Ser Trp Gly Ser Pro Leu Ile Glu Tyr Gln 20 25 30Tyr Tyr Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe
Thr Val Pro Gly Gly Leu Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys
Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Asp Tyr65 70 75
80Phe Asn Pro Ala Thr His Glu Tyr Thr Tyr His Val Ser Pro Ile Ser
85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln 100
105303108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 303Met Gly Val Ser Asp Val Pro Arg Asp Leu
Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Ser
Ala Thr Asn Lys Thr Tyr Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr Val Pro Gly Gly
Val Arg Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr
Thr Ile Thr Val Tyr Ala Val Thr Asp Tyr65 70 75 80Phe Asn Pro Thr
Thr His Glu Tyr Ile Tyr Gln Thr Thr Pro Ile Ser 85 90 95Ile Asn Tyr
Arg Thr Glu Ile Asp Lys Pro Ser Gln 100 105304108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
304Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1
5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Asp Asp Pro Ala Ala Asn Arg
Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln 35 40 45Glu Phe Thr Val Pro Tyr Asp Leu Arg Thr Ala Thr Ile
Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala
Val Thr Asp Tyr65 70 75 80Tyr Asn Pro Ala Thr His Gln Tyr Lys Tyr
Ser Gln Ser Pro Ile Ser 85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys
Pro Ser Gln 100 105305108PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 305Met Gly Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu
Leu Ile Ser Trp Tyr Trp Glu Gly Leu Pro Tyr Gln 20 25 30Tyr Tyr Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe
Thr Val Pro Arg Asp Val Asn Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys
Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Asp Trp65 70 75
80Tyr Asn Pro Asp Thr His Glu Tyr Ile Tyr His Thr Ile Pro Ile Ser
85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln 100
105306108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 306Met Gly Val Ser Asp Val Pro Arg Asp Leu
Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Ser
Ala Pro Trp Arg Thr Tyr Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr Val Pro Tyr Asp
Val Tyr Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr
Thr Ile Thr Val Tyr Ala Val Thr Asp Tyr65 70 75 80Leu Asn Pro Asn
Thr Leu Glu Tyr Thr Tyr Gln Arg Ile Pro Ile Ser 85 90 95Ile Asn Tyr
Arg Thr Glu Ile Asp Lys Pro Ser Gln 100 105307108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
307Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1
5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Gln Ala Ala Asn His Ser Tyr
Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln 35 40
45Glu Phe Thr Val Pro Gly Gly Val Arg Thr Ala Thr Ile Ser Gly Leu
50 55 60Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Asp
Phe65 70 75 80Phe Asn Pro Val Thr His Glu Tyr Lys Tyr Arg Thr Ile
Pro Ile Ser 85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln
100 105308108PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 308Met Gly Val Ser Asp Val Pro Arg
Asp Leu Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu Leu Ile Ser
Trp Asp Ser Gly Arg Gly Ser Tyr Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr
Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr Val Pro
Gly Gly Val Arg Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro Gly Val
Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Asp Tyr65 70 75 80Lys Pro
His Ala Asp Gly Pro His Thr Tyr His Glu Tyr Pro Ile Ser 85 90 95Ile
Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln 100
105309108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 309Met Gly Val Ser Asp Val Pro Arg Asp Leu
Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Asn
Asn Gly Gly Arg Asn Tyr Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr Val Pro Tyr Asp
Val Tyr Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr
Thr Ile Thr Val Tyr Ala Val Thr Asp Tyr65 70 75 80Lys Pro His Ala
Asp Gly Pro His Thr Tyr His Glu Tyr Pro Ile Ser 85 90 95Ile Asn Tyr
Arg Thr Glu Ile Asp Lys Pro Ser Gln 100 105310108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
310Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1
5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Val Val Pro Gln Gly Met Tyr
Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln 35 40 45Glu Phe Thr Val Pro Gly Gly Val Ser Thr Ala Thr Ile
Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala
Val Thr Asp Tyr65 70 75 80Phe Asn Pro Ala Thr His Glu Tyr Asn Tyr
His Ser Ile Pro Ile Ser 85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys
Pro Ser Gln 100 105311108PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 311Met Gly Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu
Leu Ile Ser Trp Ala Ser Asn Arg Gly Thr Tyr Gln 20 25 30Tyr Tyr Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe
Thr Val Pro Gly Gly Val Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys
Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Asp Ala65 70 75
80Phe Asn Pro Thr Thr His Glu Tyr Asn Tyr Phe Thr Thr Pro Ile Ser
85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln 100
105312108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 312Met Gly Val Ser Asp Val Pro Arg Asp Leu
Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Leu
Pro Gly Lys Leu Arg Tyr Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr Val Pro His Asp
Leu Arg Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr
Thr Ile Thr Val Tyr Ala Val Thr Asp Tyr65 70 75 80Lys Pro His Ala
Asp Gly Pro His Thr Tyr His Glu Ser Pro Ile Ser 85 90 95Ile Asn Tyr
Arg Thr Glu Ile Asp Lys Pro Ser Gln 100 105313108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
313Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1
5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro Thr Ser Arg Tyr
Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln 35 40 45Glu Phe Thr Val Pro Gly Gly Val Arg Thr Ala Thr Ile
Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala
Val Thr Asp His65 70 75 80Lys Pro His Ala Asp Gly Pro His Thr Tyr
His Glu Tyr Pro Ile Ser 85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys
Pro Ser Gln 100 105314108PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 314Met Gly Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu
Leu Ile Ser Trp Asp Ala Pro Thr Ser Arg Tyr Gln 20 25 30Tyr Tyr Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe
Thr Val Pro Gly Gly Val Arg Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys
Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Asp Tyr65 70 75
80Lys Pro His Ala Asp Gly Pro His Thr Tyr His Glu Tyr Pro Ile Ser
85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln 100
105315108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 315Met Gly Val Ser Asp Val Pro Arg Asp Leu
Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Thr
Pro Ala Asn Lys Ser Tyr Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr Val Pro His Asp
Leu Val Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr
Thr Ile Thr Val Tyr Ala Val Thr Asp His65 70 75 80Lys Pro His Ala
Asp Gly Pro His Thr Tyr His Glu Tyr Pro Ile Ser 85 90 95Ile Asn Tyr
Arg Thr Glu Ile Asp Lys Pro Ser Gln 100 105316108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
316Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1
5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro Ala Val Thr Tyr
Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln 35 40 45Glu Phe Thr Val Pro His Asp Leu Val Thr Ala Thr Ile
Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala
Val Thr Asp Tyr65 70 75 80Lys Pro His Ala Asp Gly Pro His Thr Tyr
His Glu Ser Pro Ile Ser 85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys
Pro Ser Gln 100 105317108PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 317Met Gly Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu
Leu Ile Ser Trp Thr Pro Ala Asn Lys Ser Tyr Gln 20 25 30Tyr Tyr Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe
Thr Val Pro His Asp Leu Val Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys
Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Asp His65 70 75
80Lys Pro His Ala Asp Gly Pro His Thr Tyr His Glu Ser Pro Ile Ser
85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln 100
105318103PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 318Met Gly Val Ser Asp Val Pro Arg Asp Leu
Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Thr
Pro Ala Asn Lys Ser Tyr Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr Val Pro Gly Gly
Val Arg Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr
Thr Ile Thr Val Tyr Ala Val Thr Asn Met65 70 75 80Met His Val Glu
Tyr Ser Glu Tyr Pro Ile Ser Ile Asn Tyr Arg Thr 85 90 95Glu Ile Asp
Lys Pro Ser Gln 100319108PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 319Met Gly Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu
Leu Ile Ser Trp Asp Ala Gly Ala Val Thr Tyr Gln 20 25 30Tyr Tyr Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe
Thr Val Pro His Asp Leu Val Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys
Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Asp His65 70 75
80Lys Pro His Ala Asp Gly Pro His Thr Tyr His Glu Tyr Pro Ile Ser
85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln 100
105320108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 320Met Gly Val Ser Asp Val Pro Arg Asp Leu
Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Asp
Ala Pro Thr Ser Arg Tyr Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr Val Pro Gly Gly
Leu Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr
Thr Ile Thr Val Tyr Ala Val Thr Asp Tyr65 70 75 80Lys Pro His Ala
Asp Gly Pro His Thr Tyr His Glu Ser Pro Ile Ser 85 90 95Ile Asn Tyr
Arg Thr Glu Ile Asp Lys Pro Ser Gln 100 105321108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
321Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1
5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro Ala Val Thr Tyr
Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln 35 40 45Glu Phe Thr Val Pro Tyr Asp Val Tyr Thr Ala Thr Ile
Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala
Val Thr Asp Tyr65 70 75 80Lys Pro His Ala Asp Gly Pro His Thr Tyr
His Glu Tyr Pro Ile Ser 85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys
Pro Ser Gln 100 105322104PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 322Met Gly Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu
Leu Ile Ser Trp Asp Ser Gly Arg Gly Ser Tyr Gln 20 25 30Tyr Tyr Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe
Thr Val Pro Tyr Asp Val Tyr Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys
Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Asp Met65 70 75
80Met His Val Glu Tyr Thr Glu His Pro Ile Ser Ile Asn Tyr Arg Thr
85 90 95Glu Ile Asp Lys Pro Ser Gln His 100323108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
323Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1
5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro Ala Val Thr Tyr
Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln 35 40 45Glu Phe Thr Val Pro Gly Gly Val Arg Thr Ala Thr Ile
Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala
Val Thr Asp Tyr65 70 75 80Lys Pro His Ala Asp Gly Pro His Thr Tyr
His Glu Ser Pro Ile Ser 85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys
Pro Ser Gln 100 105324108PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 324Met Gly Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu
Leu Ile Ser Trp Asp Ala Pro Thr Ser Arg Tyr Gln 20 25 30Tyr Tyr Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe
Thr Val Pro His Asp Leu Val Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys
Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Asp Tyr65 70 75
80Lys Pro His Ala Asp Gly Pro His Thr Tyr His Glu Ser Pro Ile Ser
85 90 95Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln 100
105325108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 325Met Gly Val Ser Asp Val Pro Arg Asp Leu
Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Asp
Ala Gly Ala Val Thr Tyr Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr Val Pro Gly Gly
Val Arg Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr
Thr Ile Thr Val Tyr Ala Val Thr Asp Tyr65 70 75 80Lys Pro His Ala
Asp Gly Pro His Thr Tyr His Glu Ser Pro Ile Ser 85 90 95Ile Asn Tyr
Arg Thr Glu Ile Asp Lys Pro Ser Gln 100 105326103PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
326Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr1
5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro Thr Ser Arg Tyr
Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro
Val Gln 35 40 45Glu Phe Thr Val Pro His Asp Leu Val Thr Ala Thr Ile
Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala
Val Thr Asn Met65 70 75 80Met His Val Glu Tyr Ser Glu Tyr Pro Ile
Ser Ile Asn Tyr Arg Thr 85 90 95Glu Ile Asp Lys Pro Ser Gln
100327108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 327Met Gly Val Ser Asp Val Pro Arg Asp Leu
Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu Leu Ile Ser Trp Asp
Ala Gly Ala Val Thr Tyr Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr Val Pro Gly Gly
Val Arg Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro Gly Val Asp Tyr
Thr Ile Thr Val Tyr Ala Val Thr Asp Tyr65 70 75 80Lys Pro His Ala
Asp Gly Pro His Thr Tyr His Glu Tyr Pro Ile Ser 85 90 95Ile Asn Tyr
Arg Thr Glu Ile Asp Lys Pro Ser Gln 100 105328315DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
328atgggagttt ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc
caccagcctg 60ctgatcagct ggtctgcgcg tctgaaagtt gcgcgatatt accgcatcac
ttacggcgaa 120acaggaggca atagccctgt ccaggagttc actgtgccta
aaaacgttta cacagctacc 180atcagcggcc ttaaacctgg cgttgattat
accatcactg tgtatgctgt cactaggttc 240cgcgactacc agccaatttc
cattaattac cgcacagaaa ttgacaaacc atgccagcac 300caccaccacc accac
315329342DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 329atgggagttt ctgatgtgcc gcgcgacctg
gaagtggttg ctgccacccc caccagcctg 60ctgatcagct ggcaggttcc gcgtccgatg
taccaatatt accgcatcac ttacggcgaa 120acaggaggca atagccctgt
ccaggagttc actgtgcctt acgacgttta cacagctacc 180atcagcggcc
ttaaacctgg cgttgattat accatcactg tgtatgctgt cactgactct
240tacaacccgg ctactcatga atacaaatac catcagactc caatttccat
taattaccgc 300acagaaattg acaaaccatc ccagcaccat caccaccacc ac
342330342DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 330atgggagttt ctgatgtgcc gcgcgacctg
gaagtggttg ctgccacccc caccagcctg 60ctgatcagct ggcaggttcc gcgtccgatg
taccaatatt accgcatcac ttacggcgaa 120acaggaggca atagccctgt
ccaggagttc actgtgcctt acgacgttca tacagctacc 180atcagcggcc
ttaaacctgg cgttgattat accatcactg tgtatgctgt cactgactct
240tacaacccgg ctactcatga atacaaatac catcagactc caatttccat
taattaccgc 300acagaaattg acaaaccatc ccagcaccat caccaccacc ac
342331342DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 331atgggagttt ctgatgtgcc gcgcgacctg
gaagtggttg ctgccacccc caccagcctg 60ctgatcagct ggcaggttcc gcgtccgatg
taccaatatt accgcatcac ttacggcgaa 120acaggaggca atagccctgt
ccaggagttc actgtgcctt acgacctgac tacagctacc 180atcagcggcc
ttaaacctgg cgttgattat accatcactg tgtatgctgt cactgactct
240tacaacccgg ctactcatga atacaaatac catcagactc caatttccat
taattaccgc 300acagaaattg acaaaccatc ccagcaccat caccaccacc ac
342332342DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 332atgggagttt ctgatgtgcc gcgcgacctg
gaagtggttg ctgccacccc caccagcctg 60ctgatcagct gggaagctaa cccttctcgt
tatcaatatt accgcatcac ttacggcgaa 120acaggaggca atagccctgt
ccaggagttc actgtgcctc atgacctgaa cacagctacc 180atcagcggcc
ttaaacctgg cgttgattat accatcactg tgtatgctgt cactgactct
240tacaacccgg ctactcatga atacaaatac catcagactc caatttccat
taattaccgc 300acagaaattg acaaaccatc ccagcaccat caccaccacc ac
342333342DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 333atgggagttt ctgatgtgcc gcgcgacctg
gaagtggttg ctgccacccc caccagcctg 60ctgatcagct ggtacccagg atctcgcacc
taccaatatt accgcatcac ttacggcgaa 120acaggaggca atagccctgt
ccaggagttc actgtgcctg gtggtgttcg tacagctacc 180atcagcggcc
ttaaacctgg cgttgattat accatcactg tgtatgctgt cactgactac
240taccatccgg ctacttacga acatgaatac catgctcatc caatttccat
taattaccgc 300acagaaattg acaaaccatc ccagcaccat caccaccacc ac
342334342DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 334atgggagttt ctgatgtgcc gcgcgacctg
gaagtggttg ctgccacccc caccagcctg 60ctgatcagct ggacccctgc taataaatct
taccaatatt accgcatcac ttacggcgaa 120acaggaggca atagccctgt
ccaggagttc actgtgcctt acgacggtac tacagctacc 180atcagcggcc
ttaaacctgg cgttgattat accatcactg tgtatgctgt cactgaccat
240aaaccgcatg ctgacggtcc gcatacttac catgaatacc caatttccat
taattaccgc 300acagaaattg acaaaccatc ccagcaccat caccaccacc ac
342335342DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 335atgggagttt ctgatgtgcc gcgcgacctg
gaagtggttg ctgccacccc caccagcctg 60ctgatcagct ggcaggttcc gcgtccgatg
taccaatatt accgcatcac ttacggcgaa 120acaggaggca atagccctgt
ccaggagttc actgtgcctc atgacgttta cacagctacc 180atcagcggcc
ttaaacctgg cgttgattat accatcactg tgtatgctgt cactgactct
240tacaacccgg ctactcatga atacaaatac catcagactc caatttccat
taattaccgc 300acagaaattg acaaaccatc ccagcaccat caccaccacc ac
342336342DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 336atgggagttt ctgatgtgcc gcgcgacctg
gaagtggttg ctgccacccc caccagcctg 60ctgatcagct ggcaggttcc gcgtccgatg
taccaatatt accgcatcac ttacggcgaa 120acaggaggca atagccctgt
ccaggagttc actgtgcctg gtcaggttta cacagctacc 180atcagcggcc
ttaaacctgg cgttgattat accatcactg tgtatgctgt cactgactct
240tacaacccgg ctactcatga atacaaatac catcagactc caatttccat
taattaccgc 300acagaaattg acaaaccatc ccagcaccat caccaccacc ac
342337342DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 337atgggagttt ctgatgtgcc gcgcgacctg
gaagtggttg ctgccacccc caccagcctg 60ctgatcagct ggcaggttcc gcgtccgatg
taccaatatt accgcatcac ttacggcgaa 120acaggaggca atagccctgt
ccaggagttc actgtgcctt acgacgttac tacagctacc 180atcagcggcc
ttaaacctgg cgttgattat accatcactg tgtatgctgt cactgactct
240tacaacccgg ctactcatga atacaaatac catcagactc caatttccat
taattaccgc 300acagaaattg acaaaccatc ccagcaccat caccaccacc ac
342338342DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 338atgggagttt ctgatgtgcc gcgcgacctg
gaagtggttg ctgccacccc caccagcctg 60ctgatcagct ggcaggttcc gcgtccgatg
taccaatatt accgcatcac ttacggcgaa 120acaggaggca atagccctgt
ccaggagttc actgtgcctc atgacctgcg tacagctacc 180atcagcggcc
ttaaacctgg cgttgattat accatcactg tgtatgctgt cactgactct
240tacaacccgg ctactcatga atacaaatac catcagactc caatttccat
taattaccgc 300acagaaattg acaaaccatc ccagcaccat caccaccacc ac
342339342DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 339atgggagttt ctgatgtgcc gcgcgacctg
gaagtggttg ctgccacccc caccagcctg 60ctgatcagct ggcaggttcc gcgtccgatg
taccaatatt accgcatcac ttacggcgaa 120acaggaggca atagccctgt
ccaggagttc actgtgcctt acgacctggt tacagctacc 180atcagcggcc
ttaaacctgg cgttgattat accatcactg tgtatgctgt cactgaccat
240aaaccgcatg ctgacggtcc gcatacttac catgaatctc caatttccat
taattaccgc 300acagaaattg acaaaccatc ccagcaccat caccaccacc ac
342340327DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 340atgggagttt ctgatgtgcc gcgcgacctg
gaagtggttg ctgccacccc caccagcctg 60ctgatcagct ggttgccggg caagctgagg
taccaatatt accgcatcac ttacggcgaa 120acaggaggca atagccctgt
ccaggagttc actgtgcctc atgacctgcg tacagctacc 180atcagcggcc
ttaaacctgg cgttgattat accatcactg tgtatgctgt cactaacatg
240atgcatgttg aatactctga atacccaatt tccattaatt accgcacaga
aattgacaaa 300ccatcccagc accatcacca ccaccac 327341327DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
341atgggagttt ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc
caccagcctg 60ctgatcagct gggtggccgg ggcggaggac taccaatatt accgcatcac
ttacggcgaa 120acaggaggca atagccctgt ccaggagttc actgtgcctc
atgacctggt tacagctacc 180atcagcggcc ttaaacctgg cgttgattat
accatcactg tgtatgctgt cactgacatg 240atgcatgttg aatacactga
acatccaatt tccattaatt accgcacaga aattgacaaa 300ccatcccagc
accatcacca ccaccac 327342342DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 342atgggagttt
ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc caccagcctg 60ctgatcagct
ggcaggttcc gcgtccgatg taccaatatt accgcatcac ttacggcgaa
120acaggaggca atagccctgt ccaggagttc actgtgcctg gtatggttca
cacagctacc 180atcagcggcc ttaaacctgg cgttgattat accatcactg
tgtatgctgt cactgaccat 240aaaccgcatg ctgacggtcc gcatacttac
catgaatctc caatttccat taattaccgc 300acagaaattg acaaaccatc
ccagcaccat caccaccacc ac 342343342DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 343atgggagttt
ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc caccagcctg 60ctgatcagct
ggttcgtgac gcacgtcgcc taccaatatt accgcatcac ttacggcgaa
120acaggaggca atagccctgt ccaggagttc actgtgcctg gtggtctgtc
cacagctacc 180atcagcggcc ttaaacctgg cgttgattat accatcactg
tgtatgctgt cactgactat 240aaaccgcatg ctgacggtcc gcatacttac
catgaatctc caatttccat taattaccgc 300acagaaattg acaaaccatc
ccagcaccat caccaccacc ac 342344342DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 344atgggagttt
ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc caccagcctg 60ctgatcagct
gggagacgga gagcaacgcg taccaatatt accgcatcac ttacggcgaa
120acaggaggca atagccctgt ccaggagttc actgtgcctg gtcagatcta
cacagctacc 180atcagcggcc ttaaacctgg cgttgattat accatcactg
tgtatgctgt cactgactat 240aaaccgcatg ctgacggtcc gcatacttac
catgaatctc caatttccat taattaccgc 300acagaaattg acaaaccatc
ccagcaccat caccaccacc ac 342345342DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 345atgggagttt
ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc caccagcctg 60ctgatcagct
ggatgacgtc gccctcggtg taccaatatt accgcatcac ttacggcgaa
120acaggaggca atagccctgt ccaggagttc actgtgcctg gtccggttca
gacagctacc 180atcagcggcc ttaaacctgg cgttgattat accatcactg
tgtatgctgt cactgactac 240aaagaacatc agcatgctcc gcatcagtac
actgctcatc caatttccat taattaccgc 300acagaaattg acaaaccatc
ccagcaccat caccaccacc ac 342346342DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 346atgggagttt
ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc caccagcctg 60ctgatcagct
gggattcagg acgaggttcc tatcaatatt accgcatcac ttacggcgaa
120acaggaggca atagccctgt ccaggagttc actgtgcctg gtccggttca
tacagctacc 180atcagcggcc ttaaacctgg cgttgattat accatcactg
tgtatgctgt cactgaccat 240aaaccgcatg ctgacggtcc gcatacttac
catgaatctc caatttccat taattaccgc 300acagaaattg acaaaccatc
ccagcaccat caccaccacc ac 342347342DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 347atgggagttt
ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc caccagcctg 60ctgatcagct
ggtcaacagg tcgcacaact tatcaatatt accgcatcac ttacggcgaa
120acaggaggca atagccctgt ccaggagttc actgtgcctc atgacctgga
cacagctacc 180atcagcggcc ttaaacctgg cgttgattat accatcactg
tgtatgctgt cactgactct 240tacaacccgg ctactcatga atacaaatac
catcagactc caatttccat taattaccgc 300acagaaattg acaaaccatc
ccagcaccat caccaccacc ac 342348327DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 348atgggagttt
ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc caccagcctg 60ctgatcagct
ggcaggttcc gcgtccgatg taccaatatt accgcatcac ttacggcgaa
120acaggaggca atagccctgt ccaggagttc actgtgcctt acgacgttat
cacagctacc 180atcagcggcc ttaaacctgg cgttgattat accatcactg
tgtatgctgt cactgacatg 240atgcatgttg aatacgctga atacccaatt
tccattaatt accgcacaga aattgacaaa 300ccatcccagc accatcacca ccaccac
327349342DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 349atgggagttt ctgatgtgcc gcgcgacctg
gaagtggttg ctgccacccc caccagcctg 60ctgatcagct ggcaggttcc gcgtccgatg
taccaatatt accgcatcac ttacggcgaa 120acaggaggca atagccctgt
ccaggagttc actgtgcctg gtcaggttcc aacagctacc 180atcagcggcc
ttaaacctgg cgttgattat accatcactg tgtatgctgt cactgactct
240tacaacccgg ctactcatga atacaaatac catcagactc caatttccat
taattaccgc 300acagaaattg acaaaccatc ccagcaccat caccaccacc ac
342350327DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 350atgggagttt ctgatgtgcc gcgcgacctg
gaagtggttg ctgccacccc caccagcctg 60ctgatcagct ggggctacca aagtggcggc
tatacctatt accgcatcac ttacggcgaa 120acaggaggca atagccctgt
ccaggagttc actgtgcctc atgacctgcg tacagctacc 180atcagcggcc
ttaaacctgg cgttgattat accatcactg tgtatgctgt cactgactac
240gcttacaaag aataccagga acatccaatt tccattaatt accgcacaga
aattgacaaa 300ccatcccagc accatcacca ccaccac 327351327DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
351atgggagttt ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc
caccagcctg 60ctgatcagct ggtggatcgg catcccggtg taccaatatt accgcatcac
ttacggcgaa 120acaggaggca atagccctgt ccaggagttc actgtgcctt
acgacggtaa aacagctacc 180atcagcggcc ttaaacctgg cgttgattat
accatcactg tgtatgctgt cactgacatg 240atgcatgttg aatacgctga
atacccaatt tccattaatt accgcacaga aattgacaaa 300ccatcccagc
accatcacca ccaccac 327352342DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 352atgggagttt
ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc caccagcctg 60ctgatcagct
ggtctaaagg ttcaaaatct taccaatatt accgcatcac ttacggcgaa
120acaggaggca atagccctgt ccaggagttc actgtgcctt accatgttta
cacagctacc 180atcagcggcc ttaaacctgg cgttgattat accatcactg
tgtatgctgt cactgactat 240tataacccgg ctacttacga atacatatac
cttacgactc caatttccat taattaccgc 300acagaaattg acaaaccatc
ccagcaccat caccaccacc ac 342353342DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 353atgggagttt
ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc caccagcctg 60ctgatcagct
ggaatcccgg ctccaaaagc taccaatatt accgcatcac ttacggcgaa
120acaggaggca atagccctgt ccaggagttc actgtgcctg gtggtgttcg
tacagctacc 180atcagcggcc ttaaacctgg cgttgattat accatcactg
tgtatgctgt cactgacttt 240tacaatccgg atactcatga atacctatac
aatcaatatc caatttccat taattaccgc 300acagaaattg acaaaccatc
ccagcaccat caccaccacc ac 342354342DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 354atgggagttt
ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc caccagcctg 60ctgatcagct
ggcaacccgg caccacacat tatcaatatt accgcatcac ttacggcgaa
120acaggaggca atagccctgt ccaggagttc actgtgcctt acgacctgat
gacagctacc 180atcagcggcc ttaaacctgg cgttgattat accatcactg
tgtatgctgt cactgactat 240tacaacccga atacttatga gtatatatac
ttgacgactc caatttccat taattaccgc 300acagaaattg acaaaccatc
ccagcaccat caccaccacc ac 342355342DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 355atgggagttt
ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc caccagcctg 60ctgatcagct
gggccatcgg caccatcgtc taccaatatt accgcatcac ttacggcgaa
120acaggaggca atagccctgt ccaggagttc actgtgcctg ctggtgttta
cacagctacc 180atcagcggcc ttaaacctgg cgttgattat accatcactg
tgtatgctgt cactgactat 240tacgactggg ctactcatga atacaattac
cacaccgctc caatttccat taattaccgc 300acagaaattg acaaaccatc
ccagcaccat caccaccacc ac 342356342DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 356atgggagttt
ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc caccagcctg 60ctgatcagct
ggacttataa tgatggcagc tatcaatatt accgcatcac ttacggcgaa
120acaggaggca atagccctgt ccaggagttc actgtgcctt acgctgttta
cacagctacc 180atcagcggcc ttaaacctgg cgttgattat accatcactg
tgtatgctgt cactgacttt 240tacaatccgg ctacatatga atacatatat
cacacgacac caatttccat taattaccgc 300acagaaattg acaaaccatc
ccagcaccat caccaccacc ac 342357342DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 357atgggagttt
ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc caccagcctg 60ctgatcagct
gggtctccct cgtgggcttc taccaatatt accgcatcac ttacggcgaa
120acaggaggca atagccctgt ccaggagttc actgtgcctg gtggtgttca
tacagctacc 180atcagcggcc ttaaacctgg cgttgattat accatcactg
tgtatgctgt cactgactat 240aaaccgcatg ctgacggtcc gcatacttac
catgaatctc caatttccat taattaccgc 300acagaaattg acaaaccatc
ccagcaccat caccaccacc ac 342358327DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 358atgggagttt
ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc caccagcctg 60ctgatcagct
gggcctcgag gaaggaggtc taccaatatt accgcatcac ttacggcgaa
120acaggaggca atagccctgt ccaggagttc actgtgcctg gttggttgaa
cacagctacc 180atcagcggcc ttaaacctgg cgttgattat accatcactg
tgtatgctgt cactgactac 240atgcatgttg aatacgctga atacccaatt
tccattaatt accgcacaga aattgacaaa 300ccatcccagc accatcacca ccaccac
327359342DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 359atgggagttt ctgatgtgcc gcgcgacctg
gaagtggttg ctgccacccc caccagcctg 60ctgatcagct ggttggcgcc cttctggcgg
taccaatatt accgcatcac ttacggcgaa 120acaggaggca atagccctgt
ccaggagttc actgtgcctg gtggtgttcg tacagctacc 180atcagcggcc
ttaaacctgg cgttgattat accatcactg tgtatgctgt cactgactat
240aaaccgcatg ctgacggtcc gcatacttac catgaatctc caatttccat
taattaccgc 300acagaaattg acaaaccatc ccagcaccat caccaccacc ac
342360342DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 360atgggagttt ctgatgtgcc gcgcgacctg
gaagtggttg ctgccacccc caccagcctg 60ctgatcagct ggacaccacc aggacatcaa
catcaatatt accgcatcac ttacggcgaa 120acaggaggca atagccctgt
ccaggagttc actgtgcctg gtcaggttac tacagctacc 180atcagcggcc
ttaaacctgg cgttgattat accatcactg tgtatgctgt cactgactat
240tacaacccag ctactcacta ttacacttat tatacgactc caatttccat
taattaccgc 300acagaaattg acaaaccatc ccagcaccat caccaccacc ac
342361342DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 361atgggagttt ctgatgtgcc gcgcgacctg
gaagtggttg ctgccacccc caccagcctg 60ctgatcagct gggagtcggg gtccaggacg
taccaatatt accgcatcac ttacggcgaa 120acaggaggca atagccctgt
ccaggagttc actgtgcctg gtggtgttca tacagctacc 180atcagcggcc
ttaaaactgg cgttgattat accatcactg tgtatgctgt cactgactat
240aaaccgcatg ctgacggtcc gcatacttac catgaatatc caatttccat
taattaccgc 300acagaaattg acaaaccatc ccagcaccat caccaccacc ac
342362342DNAArtificial SequenceDescription of Artificial
Sequence
Synthetic polynucleotide 362atgggagttt ctgatgtgcc gcgcgacctg
gaagtggttg ctgccacccc caccagcctg 60ctgatcagct gggagaggac ctccacccac
taccaatatt accgcatcac ttacggcgaa 120acaggaggca atagccctgt
ccaggagttc actgtgcctg gtcgtgttta cacagctacc 180atcagcggcc
ttaaacctgg cgttgattat accatcactg tgtatgctgt cactgactac
240tacaacccgg ctactcatga atacaaatac catcagactc caatttccat
taattaccgc 300acagaaattg acaaaccatc ccagcaccat caccaccacc ac
342363342DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 363atgggagttt ctgatgtgcc gcgcgacctg
gaagtggttg ctgccacccc caccagcctg 60ctgatcagct ggaatgctcg caccgacgct
tatcaatatt accgcatcac ttacggcgaa 120acaggaggca atagccctgt
ccaggagttc actgtgcctc gtgacctgga aacagctacc 180atcagcggcc
ttaaacctgg cgttgattat accatcactg tgtatgctgt cactgactat
240aaacctcatg cggacggacc gcatacttac caagagtcgc caatttccat
taattaccgc 300acagaaattg acaaaccatc ccagcaccat caccaccacc ac
342364342DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 364atgggagttt ctgatgtgcc gcgcgacctg
gaagtggttg ctgccacccc caccagcctg 60ctgatcagct ggcaggtgag cgcgttccgg
taccaatatt accgcatcac ttacggcgaa 120acaggaggca atagccctgt
ccaggagttc actgtgcctg gtatggtttc tacagctacc 180atcagcggcc
ttaaacctgg cgttgattat accatcactg tgtatgctgt cactgactat
240aaaccgcatg ctgacggtcc gcatacttac tctgaatacc caatttccat
taattaccgc 300acagaaattg acaaaccatc ccagcaccat caccaccacc ac
342365342DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 365atgggagttt ctgatgtgcc gcgcgacctg
gaagtggttg ctgccacccc caccagcctg 60ctgatcagct gggtgctggg caggagggtg
taccaatatt accgcatcac ttacggcgaa 120acaggaggca atagccctgt
ccaggagttc actgtgcctg gtgctgttta cacagctacc 180atcagcggcc
ttaaacctgg cgttgattat accatcactg tgtatgctgt cactgactat
240ttcaacccag ctacccatga ataccaatac gagcttactc caatttccat
taattaccgc 300acagaaattg acaaaccatc ccagcaccat caccaccacc ac
342366342DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 366atgggagttt ctgatgtgcc gcgcgacctg
gaagtggttg ctgccacccc caccagcctg 60ctgatcagct ggactccacc caattctggt
cataattatt accgcatcac ttacggcgaa 120acaggaggca atagccctgt
ccaggagttc actgtgcctc atgacctgac tacagctacc 180atcagcggcc
ttaaacctgg cgttgattat accatcactg tgtatgctgt cactgactat
240tacaacccga atacctatga atacacatat caattcactc caatttccat
taattaccgc 300acagaaattg acaaaccatc ccagcaccat caccaccacc ac
342367342DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 367atgggagttt ctgatgtgcc gcgcgacctg
gaagtggttg ctgccacccc caccagcctg 60ctgatcagct gggtcgtccc gaactggatg
taccaatatt accgcatcac ttacggcgaa 120acaggaggca atagccctgt
ccaggagttc actgtgcctg gtatgctgga aacagctacc 180atcagcggcc
ttaaacctgg cgttgattat accatcactg tgtatgctgt cactgactat
240tataacccga ctacgtatga atacacatac tttacctatc caatttccat
taattaccgc 300acagaaattg acaaaccatc ccagcaccat caccaccacc ac
342368342DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 368atgggagttt ctgatgtgcc gcgcgacctg
gaagtggttg ctgccacccc caccagcctg 60ctgatcagct ggtccggcgg gttcatgcgg
taccaatatt accgcatcac ttacggcgaa 120acaggaggca atagccctgt
ccaggagttc actgtgcctg gtcaggttta cacagctacc 180atcagcggcc
ttaaacctgg cgttgattat accatcactg tgtatgctgt cactgactat
240aaaccgcatg ctgacggtcc gcatacttac catgaatctc caatttccat
taattaccgc 300acagaaattg acaaaccatc ccagcaccat caccaccacc ac
342369342DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 369atgggagttt ctgatgtgcc gcgcgacctg
gaagtggttg ctgccacccc caccagcctg 60ctgatcagct gggattccga aggtccttct
tatcaatatt accgcatcac ttacggcgaa 120aaaggaggca atagccctgt
ccaggagttc actgtgcctt acgctgttta cacagctacc 180atcagcggcc
ttaaacctgg cgttgattat accatcactg tgtatgctgt cactgactat
240tacaacccga gaacgcatga attatttttc cagcaatatc caatttccat
taattaccgc 300acagaaattg acaaaccatc ccagcaccat caccaccacc ac
342370342DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 370atgggagttt ctgatgtgcc gcgcgacctg
gaagtggttg ctgccacccc caccagcctg 60ctgatcagct ggcaggttcc gcgtccgatg
taccaatatt accgcatcac ttacggcgaa 120acaggaggca atagccctgt
ccaggagttc actgtgcctc atgacctgcg tacagctacc 180atcagcggcc
ttaaacctgg cgttgattat accatcactg tgtatgctgt cactgactat
240tacgacccga catctaatct gtacaattac aaccagactc caatttccat
taattaccgc 300acagaaattg acaaaccatc ccagcaccat caccaccacc ac
342371342DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 371atgggagttt ctgatgtgcc gcgcgacctg
gaagtggttg ctgccacccc caccagcctg 60ctgatcagct ggcaggtggg ctcggtggtg
taccaatatt accgcatcac ttacggcgaa 120acaggaggca atagccctgt
ccaggagttc actgtgcctc gtgacgttct gacagctacc 180atcagcggcc
ttaaacctgg cgttgattat accatcactg tgtatgctgt cactgactat
240aaaccgaagc ctgacggtcc acatatatac caggcagtgc caatttccat
taattaccgc 300acagaaattg acaaaccatc ccagcaccat caccaccacc ac
342372342DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 372atgggagttt ctgatgtgcc gcgcgacctg
gaagtggttg ctgccacccc caccagcctg 60ctgatcagct ggaaccctgc ttctaaagac
tatcaatatt accgcatcac ttacggcgaa 120acaggaggca atagccctgt
ccaggagttc actgtgcctg gtcaggttcc gacagctacc 180atcagcggcc
ttaaacctgg cgttgattat accatcactg tgtatgctgt cactgacttt
240tacaacccgg ctactcatga gtataaatat gactcgactc caatttccat
taattaccgc 300acagaaattg acaaaccatc ccagcaccat caccaccacc ac
342373342DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 373atgggagttt ctgatgtgcc gcgcgacctg
gaagtggttg ctgccacccc caccagcctg 60ctgatcagct ggagatcatc agcaaccgcc
taccaatatt accgcatcac ttacggcgaa 120acaggaggca atagccctgt
ccaggagttc actgtgcctg gtcgtgttta cacagctacc 180atcagcggcc
ttaaacctgg cgttgattat accatcactg tgtatgctgt cactgacttt
240ttcaactggg ccactcatga gtacatatac cactcaactc caatttccat
taattaccgc 300acagaaattg acaaaccatc ccagcaccat caccaccacc ac
342374342DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 374atgggagttt ctgatgtgcc gcgcgacctg
gaagtggttg ctgccacccc caccagcctg 60ctgatcagct ggcattccgg tccacgagaa
tatcaatatt accgcatcac ttacggcgaa 120acaggaggca atagccctgt
ccaggagttc actgtgcctg gtcaggttta cacagctacc 180atcagcggcc
ttaaacctgg cgttgattat accatcactg tgtatgctgt cactgacttt
240ttcaacccga ttacacatta ctattactac gagctgactc caatttccat
taattaccgc 300acagaaattg acaaaccatc ccagcaccat caccaccacc ac
342375342DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 375atgggagttt ctgatgtgcc gcgcgacctg
gaagtggttg ctgccacccc caccagcctg 60ctgatcagct ggacggtggg cctgagcgtg
taccaatatt accgcatcac ttacggcgaa 120acaggaggca atagccctgt
ccaggagttc actgtgcctg gtatggtttc tacagctacc 180atcagcggcc
ttaaacctag cgttgattat accatcactg tgtatgctgt cactgactat
240aaaccgcatg ctgacggtcc gcatacttac catgaatatc caatttccat
taattaccgc 300acagaaattg acaaaccatc ccagcaccat caccaccacc ac
342376342DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 376atgggagttt ctgatgtgcc gcgcgacctg
gaagtggttg ctgccacccc caccagcctg 60ctgatcagct ggggggggca ccgggcggtg
taccaatatt accgcatcac ttacggcgaa 120acaggaggca atagccctgt
ccaggagttc actgtgcctg gtgctgttta cacagctacc 180atcagcggcc
ttaaacctgg cgttgattat accatcactg tgtatgctgt cactgactac
240tacaacccgg atactcatga atacaaatac catcaatatc caatttccat
taattaccgc 300acagaaattg acaaaccatc ccagcaccat caccaccacc ac
342377327DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 377atgggagttt ctgatgtgcc gcgcgacctg
gaagtggttt ctgccacccc caccagcctg 60ctgatcagct ggcaggttcc gcgtccgatg
taccaatatt accgcatcac ttacggcgaa 120acaggaggca atagccctgt
ccaggagttc actgtgcctg gtggtgttcg tacagctacc 180atcagcggcc
ttaaacctgg cgttgattat accatcactg tgtatgctgt cactgactac
240tggttcaagg aataccgtga agacccaatt tccattaatt accgcacaga
aattgacaaa 300ccatcccagc accatcacca ccaccac 327378342DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
378atgggagttt ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc
caccagcctg 60ctgatcagct ggagcgtcgg gggcatgatc taccaatatt accgcatcac
ttacggcgaa 120acaggaggca atagccctgt ccaggagttc actgtgcctg
gtatggttac tacagctacc 180atcagcggcc ttaaacctgg cgttgattat
accatcactg tgtatgctgt cactgactac 240tacaacccgg ctactcatga
atacaaatac catcagactc caatttccat taattaccgc 300acagaaattg
acaaaccatc ccagcaccat caccaccacc ac 342379342DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
379atgggagttt ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc
caccagcctg 60ctgatcagct ggtgggcccc cgtcgaccgg taccaatatt accgcatcac
ttacggcgaa 120acaggaggca atagccctgt ccaggagttc actgtgcctc
gtgacgttta cacagctacc 180atcagcggcc ttaaacctgg cgttgattat
accatcactg tgtatgctgt cactgactat 240aaaccgcatg ctgacggtcc
gcatacttac catgaatctc caatttccat taattaccgc 300acagaaattg
acaaaccatc ccagcaccat caccaccacc ac 342380342DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
380atgggagttt ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc
caccagcctg 60ctgatcagct ggtgggcccc cgtcgaccgg taccaatatt accgcatcac
ttacggcgaa 120acaggaggca atagccctgt ccaggagttc actgtgcctc
gtgacgttta cacagctacc 180atcagcggcc ttaaacctgg cgttgattat
accatcactg tgtatgctgt cactgactat 240aaaccgcatg ctgacggtcc
gcatacttac catgaatctc caatttccat taattaccgc 300acagaaattg
acaaaccatg ccagcaccac caccaccacc ac 342381342DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
381atgggagttt ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc
caccagcctg 60ctgatcagct ggaaagccag ctataccggc tacaactatt accgcatcac
ttacggcgaa 120acaggaggca atagccctgt ccaggagttc actgtgcctc
gtgacgttat gacagctacc 180atcagcggcc ttaaacctgg cgttgattat
accatcactg tgtatgctgt cactgacttc 240tacaatccgg atactcatca
atacacatac cgtcgcattc caatttccat taattaccgc 300acagaaattg
acaaaccatc ccagcaccat caccaccacc ac 342382342DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
382atgggagttt ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc
caccagcctg 60ctgatcagct ggtcggtggg ccaggtcttc taccaatatt accgcatcac
ttacggcgaa 120acaggaggca atagccctgt ccaggagttc actgtgcctt
acgacgttta cacagctacc 180atcagcggcc ttaaacctgg cgttgattat
accatcactg tgtatgctgt cactgactac 240tacaacccgg ctactcatga
atacaaatac catcagactc caatttccat taattaccgc 300acagaaattg
acaaaccatc ccagcaccat caccaccacc ac 342383342DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
383atgggagttt ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc
caccagcctg 60ctgatcagct ggtactctgg tgattaccat taccaatatt accgcatcac
ttacggcgaa 120acaggaggca atagccctgt ccaggagttc actgtgcctc
atgacctgga aacagctacc 180atcagcggcc ttaaacctgg cgttgattat
accatcactg tgtatgctgt cactgactat 240tacaacccgg ctactcatta
ctacaagtac gagcagacac caatttccat taattaccgc 300acagaaattg
acaaaccatc ccagcaccat caccaccacc ac 342384342DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
384atgggagttt ctgatgtgcc gcgcgacctg gaagttgttg ctgccacccc
caccagcctg 60ctgatcagct ggatcgtcca gggggggcgc taccaatatt accgcatcac
ttacggcgaa 120acaggaggca atagccctgt ccaggagttc actgtgcctg
gtatggttac tacagctacc 180atcagcggcc ttaaacctgg cgttgattat
accatcactg tgtatgctgt cactgactat 240tacaaccctt caactcatga
atacaaatac catcagactc caatttccat taattaccgc 300acagaaattg
acaaaccatc ccagcaccat caccaccacc ac 342385342DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
385atgggagttt ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc
caccagcctg 60ctgatcagct ggtcggccgt ccgctggcgg taccaatatt accgcatcac
ttacggcgaa 120acaggaggca atagccctgt ccaggagttc actgtgcctg
gtggtgttcg tacagctacc 180atcagcggcc ttaaacctgg cgttgattat
accatcactg tgtatgctgt cactgacttt 240tacaacccgc gtactcatgt
atacatatac gatcagttcc caatttccat taattaccgc 300acagaaattg
acaaaccatc ccagcaccat caccaccacc ac 342386342DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
386atgggagttt ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc
caccagcctg 60ctgatcagct ggagggccag gcgcttgcag taccaatatt accgcatcac
ttacggcgaa 120acaggaggca atagccctgt ccaggagttc actgtgcctg
gtatggttac tacagctacc 180atcagcggcc ttaaacctgg cgttgattat
accatcactg tgtatgctgt cactgacttt 240tacaacccgg ctactatgga
gtacacatat cagcggactc caatttccat taattaccgc 300acagaaattg
acaaaccatc ccagcaccat caccaccacc ac 342387342DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
387atgggagttt ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc
caccagcctg 60ctgatcagct ggttgcagcc cctctggagg taccaatatt accgcatcac
ttacggcgaa 120acaggaggca atagccctgt ccaggagttc actgtgcctg
gtggtctgga cacagctacc 180atcagcggac ttaaacctgg cgttgattat
accatcactg tgtatgctgt cactgactat 240aaaccgcatg ttgacggtcc
ccatgcttac catgaatatc caatttccat taattaccgc 300acagaaattg
acaaaccatc ccagcaccat caccaccacc ac 342388342DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
388atgggagttt ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc
caccagcctg 60ctgatcagct gggacgcctc ccaggggaac taccaatatt accgcatcac
ttacggcgaa 120acaggaggca atagccctgt ccaggagttc actgtgcctg
gtgctgttaa aacagctacc 180atcagcggcc ttaaacctgg cgttgattat
accatcactg tgtatgctgt cactgacttt 240ttcaacccgg ctactcatga
atacatatac catacgactc caatttccat taattaccgc 300acagaaattg
acaaaccatc ccagcaccat caccaccacc ac 342389342DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
389atgggagttt ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc
caccagcctg 60ctgatcagct ggtgcctcga cgggcagttg taccaatatt accgcatcac
ttacggcgaa 120acaggaggca atagccctgt ccaggagttc actgtgcctg
gttctatcgt tacagctacc 180atcagcggcc ttaaacctgg cgttgattat
accatcactg tgtatgctgt cactgactgg 240tacaacctcg cgactcatga
atacaactac cgtgtgactc caatttccat taattaccgc 300acagaaattg
acaaaccatc ccagcaccat caccaccacc ac 342390342DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
390atgggagttt ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc
caccagcctg 60ctgatcagct gggacacttc aggtgcttca tatcaatatt accgcatcac
ttacggcgaa 120acaggaggca atagccctgt ccaggagttc actgtgcctt
actctgttta cacagctacc 180atcagcggcc ttaaacctgg cgttgattat
accatcactg tgtatgctgt cactgactat 240tacgaccctg attcgcatta
ttacaactac aatatggttc caatttccat taattaccgc 300acagaaattg
acaaaccatc ccagcaccat caccaccacc ac 342391342DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
391atgggagttt ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc
caccagcctg 60ctgatcagct gggattctgg taatggtact tatcaatatt accgcatcac
ttacggcgaa 120acaggaggca atagccctgt ccaggagttc actgtgcctt
accgtgttta cacagctacc 180atcagcggcc ttaaacctgg cgttgattat
accatcactg tgtatgctgt cactgactat 240tacaacccgg ctactcacga
atatacatac gagctgcgtc caatttccat taattaccgc 300acagaaattg
acaaaccatc ccagcaccat caccaccacc ac 342392342DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
392atgggagttt ctgatgtgcc gcgcgacctg gaagtggttg ctaccacccc
caccagcctg 60ctgatcagct ggcggcccac cagccaggtc taccaatatt accgcatcac
ttacggcgaa 120acaggaggca atagccctgt ccaggagttc actgtgcctt
acaacgttta cacagctacc 180atcagcggcc ttaaacctgg cgttgattat
accatcactg tgtatgctgt cactgacttt 240tttaactatg ctactcacga
atacatatac cataccattc caatttccat taattaccgc 300acagaaattg
acaaaccatc ccagcaccat caccaccacc ac 342393342DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
393atgggagttt ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc
caccagcctg 60ctgatcagct ggaagtcgta cgggtcggcc taccaatatt accgcatcac
ttacggcgaa 120acaggaggca atagccctgt ccaggagttc actgtgcctg
gtgacctgca gacagctacc 180atcagcggcc ttaaacctgg cgttgattat
accatcactg tgtatgctat cactgactat 240tacaacccgg atacacatga
gtataaatac catgtgtcgc caatttccat taattaccgc 300acagaaattg
acaaaccatc ccagcaccat caccaccacc ac 342394342DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
394atgggagttt ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc
caccagcctg 60ctgatcagct ggtcgtcggt gatggggttg taccaatatt accgcatcac
ttacggcgaa 120acaggaggca atagccctgt ccaggagttc actgtgcctt
acgacgttta cacagctacc 180atcagcggcc ttaaacctgg cgttgattat
accatcactg tgtatgctgt cactgactat 240tacaaccctt ctacttatga
atacaaatac aatacgactc caatttccat taattaccgc 300acagaaattg
acaaaccatc ccagcaccat caccaccacc ac 342395342DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
395atgggagttt ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc
caccagcctg 60ctgatcagct ggcacgccgg catggcggtg taccaatatt accgcatcac
ttacggcgaa 120acaggaggca atagccctgt ccaggagttc actgtgcctg
gtgacgttct gacagctacc
180atcagcggcc ttaaacctgg cgttgattat accatcactg tgtatgctgt
cactgacttt 240ttcaatccgg ttactcatga atacatgtat catacgattc
caatttccat taattaccgc 300acagaaattg acaaaccatc ccagcaccat
caccaccacc ac 342396342DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 396atgggagttt
ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc caccagcctg 60ctgatcagct
gggtgtccgc gagggggcgg taccaatatt accgcatcac ttacggcgaa
120acaggaggca atagccctgt ccaggagttc actgtgcctg gtggtgttcg
tacagctacc 180atcagcggcc ttaaacctgg cgttgattat accatcactg
tgtatgctgt cactgactat 240tacaacctag aaacttatga atatcattac
tatcgcactc caatttccat taattaccgc 300acagaaattg acaaaccatc
ccagcaccat caccaccacc ac 342397342DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 397atgggagttt
ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc caccagcctg 60ctgatcagct
ggtggttcgg cacctcgtcc taccaatatt accgcatcac ttacggcgaa
120acaggaggca atagccctgt ccaggagttc actgtgcctg gtgacctgaa
aacagctacc 180atcagcggcc ttaaacctgg cgttgattat accatcactg
tgtatgctgt cactgactat 240tttaaccccg ttactcatga atacgaatat
catacgactc caatttccat taattaccgc 300acagaaattg acaaaccatc
ccagcaccat caccaccacc ac 342398342DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 398atgggagttt
ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc caccagcctg 60ctgatcagct
ggtccgcgac ccggaccctg taccaatatt accgaatcac ttacggcgaa
120acaggaggca atagccctgt ccaggagttc actgtgcctt acgacgttca
tacagctacc 180atcagcggcc ttaaacctgg cgttgattat accatcactg
tgtatgctgt cactgactat 240tacaacatgg ttacttatga atacaactac
catcttactc caatttccat taattaccgc 300acagaaattg acaaaccatc
ccagcaccat caccaccacc ac 342399342DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 399atgggagttt
ctgatgtgcc gcgcgacctg gaagtggttg ttgccacccc caccagcctg 60ctgatcagct
ggaccaagtt gttgggcggg taccaatatt accgcatcac ttacggcgaa
120acaggaggca atagccctgt ccaggagttc actgtgcctg gtcctgttta
cacagctacc 180atcagcggcc ttaaacctgg cgttgattat accatcactg
tgtatgctgt cactgacttt 240ttcaaccctc gtactcatga atatcaatat
cacacgactc caatttccat taattaccgc 300acagaaattg acaaaccatc
ccagcaccat caccaccacc ac 342400342DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 400atgggagttt
ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc caccagcctg 60ctgatcagct
ggagggcgtc gggcgggctg taccaatatt accgcatcac ttacggcgaa
120acaggaggca atagccctgt ccaggagttc actgtgcctg gttctgttaa
cacagctacc 180atcagcggcc ttaaacctgg cgttgattat accatcactg
tgtatgctgt cactgacttt 240tacaacccgg ctacttatga gtacatatac
cataccactc caatttccat taattaccgc 300acagaaattg acaaaccatc
ccagcaccat caccaccacc ac 342401342DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 401atgggagttt
ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc caccagcctg 60ctgatcagct
gggcggccgg gcgcgccacg taccaatatt accgcatcac ttacggcgaa
120acaggaggca atagccctgt ccaggagttc actgtgcctt acgacgttac
tacagctacc 180atcagcggcc ttaaacctgg cgttgattat accatcactg
tgtatgctgt cactgacttt 240tacaacccgg ctactcatga atactactat
gagaccacgc caatttccat taattaccgc 300acagaaattg acaaaccatc
ccagcaccat caccaccacc ac 342402342DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 402atgggagttt
ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc caccagcctg 60ctgatcagct
ggtactcgca gcccttgacg taccaatatt accgcatcac ttacggcgaa
120acaggaggca atagccctgt ccaggagttc actgtgcctc atgacgttaa
cacagctacc 180atcagcggcc ttaaacctgg cgttgattat accatcactg
tgtatgctgt cactgacttc 240tacaacccgg agacacatga atacacttac
cacctgactc caatttccat taattaccgc 300acagaaattg acaaaccatc
ccagcaccat caccaccacc ac 342403342DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 403atgggagttt
ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc caccagcctg 60ctgatcagct
ggagttctgc aacaagacct taccaatatt accgcatcac ttacggcgaa
120acaggaggca atagccctgt ccaggagttc actgtgcctg gtggtgttcg
tacagctacc 180atcagcggcc ttaaacctgg cgttgattat accatcactg
tgtatgctgt cactgacttt 240ttcaacccga ctacgcacga atactattat
catacgactc caatttccat taattaccgc 300acagaaattg acaaaccatc
ccagcaccat caccaccacc ac 342404342DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 404atgggagttt
ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc caccagcctg 60ctgatcagct
ggtccgtcga gaggtccgtg taccaatatt accgcatcac ttacggcgaa
120acaggaggca atagccctgt ccaggagttc actgtgcctg gtggtgttcg
tacagctacc 180atcagcggcc ttaaacctgg cgttgattat accatcactg
tgtatgctgt cactgactat 240tacaacccgt ctactcatga atacaattac
ctcacgactc caatttccat taattaccgc 300acagaaattg acaaaccatc
ccagcaccat caccaccacc ac 342405342DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 405atgggagttt
ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc caccagcctg 60ctgatcagct
ggcaagatac ctccagttat catcaatatt accgcatcac ttacggcgaa
120acaggaggca atagccctgt ccaggagttc actgtgcctg gtggtgttcg
tacagctacc 180atcagcggcc ttaaacctgg cgttgattat accatcactg
tgtatgctgt cactgactat 240ttcaacccgt ctacccatga atacatctac
cgtaccattc caatttccat taattaccgc 300acagaaattg acaaaccatc
ccagcaccat caccaccacc ac 342406342DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 406atgggagttt
ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc caccagcctg 60ctgatcagct
ggtctagctc tcatcgccgc tatcaatatt accgcatcac ttacggcgaa
120acaggaggca atagccctgt ccaggagttc actgtgcctg gttcggttgc
tacagctacc 180atcagcggcc ttaaacctgg cgttgattat accatcactg
tgtatgctgt cactgactat 240ttcaacccag acactcatga atacctatac
catgccaccc caatttccat taattaccgc 300acagaaattg acaaaccatc
ccagcaccat caccaccacc ac 342407342DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 407atgggagttt
ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc caccagcctg 60ctgatcagct
gggataataa ttctaactca tatcaatatt accgcatcac ttacggcgaa
120acaggaggca atagccctgt ccaggagttc actgtgcctt acgacctgcg
tacagctacc 180atcagcggcc ttaaacctgg cgttgattat accatcactg
tgtatgctgt cactgactat 240aaacctcata ctgagggtga gcatacttat
catgaatcgc caatttccat taattaccgc 300acagaaattg acaaaccatc
ccagcaccat caccaccacc ac 342408342DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 408atgggagttt
ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc caccagcctg 60ctgatcagct
ggcgcgtgtt ggtcgacatg taccaatatt accgcatcac ttacggcgaa
120acaggaggca atagccctgt ccaggagttc actgtgcctg gtggtgttct
gacagctacc 180atcagcggcc ttaaacctgg cgttgattat accatcactg
tgtatgctgt cactgactat 240aaaccgcatg ttgacgggcc gcacacctac
tatgaatctc caatttccat taattaccgc 300acagaaattg acaaaccatc
ccagcaccat caccaccacc ac 342409342DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 409atgggagttt
ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc caccagcctg 60ctgatcagct
ggatgttcgt ggggatgtcc taccaatatt accgcatcac ttacggcgaa
120acaggaggca atagccctgt ccaggagttc actgtgcctt acggtgttca
tacagctacc 180atcagcggcc ttaaacctgg cgttgattat accatcactg
tgtatgctgt cactgactat 240ttcaacccgg ctacgcatga atacatctac
catgtgactc caatttccat taattaccgc 300acagaaattg acaaaccatc
ccagcaccat caccaccacc ac 342410342DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 410atgggagttt
ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc caccagcctg 60ctgatcagct
ggacgctgca ccggaagaac taccaatatt accgcatcac ttacggcgaa
120acaggaggca atagccctgt ccaggagttc actgtgcctg gtggtgttgt
tacagctacc 180atcagcggcc ttaaacctgg cgttgattat accatcactg
tgtatgctgt cactgactat 240tacaacccgg caactcatga atacgactac
cgaacaactc caatttccat taattaccgc 300acagaaattg acaaaccatc
ccagcaccat caccaccacc ac 342411342DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 411atgggagttt
ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc caccagcctt 60ctgatcagct
ggacacaagg cagtactcat taccaatatt accgcatcac ttacggcgaa
120acaggaggca atagccctgt ccaggagttc actgtgcctg gtatggttta
cacagctacc 180atcagcggcc ttaaacctgg cgttgattat accatcactg
tgtatgctgt cactgactat 240ttcgaccgct ctactcatga gtataaatac
cgtacgactc caatttccat taattaccgc 300acagaaattg acaaaccatc
ccagcaccat caccaccacc ac 342412342DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 412atgggagttt
ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc caccagcctg 60ctgatcagct
ggcacgaacg tgacggaagt agacaatatt accgcatcac ttacggcgaa
120acaggaggca atagccctgt ccaggagttc actgtgcctg gtggtgttcg
tacagctacc 180atcagcggcc ttaaacctgg cgttgattat accatcactg
tgtatgctgt cactgactat 240tttaacccga ctacacatga atacatatat
cagacaactc caatttccat taattaccgc 300acagaaattg acaaaccatc
ccagcaccat caccaccacc ac 342413342DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 413atgggagttt
ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc caccagcctg 60ctgatcagct
gggactccgg tgaaaacaat taccaatatt accgcatcac ttacggcgaa
120acaggaggca atagccctgt ccaggagttc actgtgcctg gtggtgttcg
tacagctacc 180atcagcggcc ttaaacctgg cgttgattat accatcactg
tgtatgctgt cactgactat 240tacaacccga agactcatga atataattat
cttactattc caatttccat taattaccgc 300acagaaattg acaaaccatc
ccagcaccat caccaccacc ac 342414342DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 414atgggagttt
ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc caccagcctg 60ctgatcagct
gggggagccc cttgatcgag taccaatatt accgcatcac ttacggcgaa
120acaggaggca atagccctgt ccaggagttc actgtgcctg gtggtctgtc
tacagctacc 180atcagcggcc tcaaacctgg cgttgattat accatcactg
tgtatgctgt cactgactat 240ttcaacccgg ctactcatga atacacatac
catgtgagtc caatttccat taattaccgc 300acagaaattg acaaaccatc
ccagcaccat caccaccacc ac 342415342DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 415atgggagttt
ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc caccagcctg 60ctgatcagct
ggtctgcaac aaacaaaact taccaatatt accgcatcac ttacggcgaa
120acaggaggca atagccctgt ccaggagttc actgtgcctg gtggtgttcg
tacagctacc 180atcagcggcc ttaaacctgg cgttgattat accatcactg
tgtatgctgt cactgactat 240tttaacccga ctacacatga atacatatat
cagacaactc caatttccat taattaccgc 300acagaaattg acaaaccatc
ccagcaccat caccaccacc ac 342416342DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 416atgggagttt
ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc caccagcctg 60ctgatcagct
gggatgaccc agctgcaaac cgacaatatt accgcatcac ttacggcgaa
120acaggaggca atagccctgt ccaggagttc actgtgcctt acgacctgcg
tacagctacc 180atcagcggcc ttaaacctgg cgttgattat accatcactg
tgtatgctgt cactgactat 240tacaacccgg ctacccatca atacaaatac
tctcagagtc caatttccat taattaccgc 300acagaaattg acaaaccatc
ccagcaccat caccaccacc ac 342417342DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 417atgggagttt
ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc caccagcctg 60ctgatcagct
ggtactggga ggggctgccc taccaatatt accgcatcac ttacggcgaa
120acaggaggca atagccctgt ccaggagttc actgtgcctc gtgacgttaa
cacagctacc 180atcagcggcc ttaaacctgg cgttgattat accatcactg
tgtatgctgt cactgactgg 240tacaaccccg acacccatga gtatatatac
catacgattc caatttccat taattaccgc 300acagaaattg acaaaccatc
ccagcaccat caccaccacc ac 342418342DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 418atgggagttt
ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc caccagcctg 60ctgatcagct
ggagcgcgcc gtggcggacc taccaatatt accgcatcac ttacggcgaa
120acaggaggca atagccctgt ccaggagttc actgtgcctt acgacgttta
cacagctacc 180atcagcggcc ttaaacctgg cgttgattat accatcactg
tgtatgctgt cactgactat 240ttaaacccta acacgcttga atacacctac
cagcgcattc caatttccat taattaccgc 300acagaaattg acaaaccatc
ccagcaccat caccaccacc ac 342419342DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 419atgggagttt
ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc caccagcctg 60ctgatcagct
ggcaggcggc caaccactcg taccaatatt accgcatcac ttacggcgaa
120acaggaggca atagccctgt ccaggagttc actgtgcctg gtggtgttcg
tacagctacc 180atcagcggcc ttaaacctgg cgttgattat accatcactg
tgtatgctgt cactgacttt 240ttcaatcctg tcactcatga atacaaatac
cgtacaattc caatttccat taattaccgc 300acagaaattg acaaaccatc
ccagcaccat caccaccacc ac 342420342DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 420atgggagttt
ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc caccagcctg 60ctgatcagct
gggattcagg acgaggttcc tatcaatatt accgcatcac ttacggcgaa
120acaggaggca atagccctgt ccaggagttc actgtgcctg gtggtgttcg
tacagctacc 180atcagcggcc ttaaacctgg cgttgattat accatcactg
tgtatgctgt cactgactat 240aaaccgcacg ctgacggtcc gcacacttac
catgaatatc caatttccat taattaccgc 300acagaaattg acaaaccatc
ccagcaccat caccaccacc ac 342421342DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 421atgggagttt
ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc caccagcctg 60ctgatcagct
ggaataacgg aggacgcaat tatcaatatt accgcatcac ttacggcgaa
120acaggaggca atagccctgt ccaggagttc actgtgcctt acgacgttta
cacagctacc 180atcagcggcc ttaaacctgg cgttgattat accatcactg
tgtatgctgt cactgactat 240aaaccgcacg ctgacggtcc gcacacttac
catgaatatc caatttccat taattaccgc 300acagaaattg acaaaccatc
ccagcaccat caccaccacc ac 342422342DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 422atgggagttt
ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc caccagcctg 60ctgatcagct
gggtcgtgcc gcaggggatg taccaatatt accgcatcac ttacggcgaa
120acaggaggca atagccctgt ccaggagttc actgtgcctg gtggtgtttc
tacagctacc 180atcagcggcc ttaaacctgg cgttgattat accatcactg
tgtatgctgt cactgactat 240ttcaacccgg caacccatga atacaattat
cattcaattc caatttccat taattaccgc 300acagaaattg acaaaccatc
ccagcaccat caccaccacc ac 342423342DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 423atgggagttt
ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc caccagcctg 60ctgatcagct
gggcgagcaa ccgggggacg taccaatatt accgcatcac ttacggcgaa
120acaggaggca atagccctgt ccaggagttc actgtgcctg gtggtgtttc
tacagctacc 180atcagcggcc ttaaacctgg cgttgattat accatcactg
tgtatgctgt cactgacgct 240ttcaacccaa ctactcatga atacaattat
tttacaactc caatttccat taattaccgc 300acagaaattg acaaaccatc
ccagcaccat caccaccacc ac 342424342DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 424atgggagttt
ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc caccagcctg 60ctgatcagct
ggttgccggg caagctgagg taccaatatt accgcatcac ttacggcgaa
120acaggaggca atagccctgt ccaggagttc actgtgcctc atgacctgcg
tacagctacc 180atcagcggcc ttaaacctgg cgttgattat accatcactg
tgtatgctgt cactgactat 240aaaccgcatg ctgacggtcc gcatacttac
catgaatctc caatttccat taattaccgc 300acagaaattg acaaaccatc
ccagcaccat caccaccacc ac 342425342DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 425atgggagttt
ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc caccagcctg 60ctgatcagct
gggacgctcc aacctcccgc taccaatatt accgcatcac ttacggcgaa
120acaggaggca atagccctgt ccaggagttc actgtgcctg gtggtgttcg
tacagctacc 180atcagcggcc ttaaacctgg cgttgattat accatcactg
tgtatgctgt cactgaccat 240aaaccgcatg ctgacggtcc gcatacttac
catgaatacc caatttccat taattaccgc 300acagaaattg acaaaccatc
ccagcaccat caccaccacc ac 342426342DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 426atgggagttt
ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc caccagcctg 60ctgatcagct
gggacgctcc aacctcccgc taccaatatt accgcatcac ttacggcgaa
120acaggaggca atagccctgt ccaggagttc actgtgcctg gtggtgttcg
tacagctacc 180atcagcggcc ttaaacctgg cgttgattat accatcactg
tgtatgctgt cactgactat 240aaaccgcacg ctgacggtcc gcacacttac
catgaatatc caatttccat taattaccgc 300acagaaattg acaaaccatc
ccagcaccat caccaccacc ac 342427342DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 427atgggagttt
ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc caccagcctg 60ctgatcagct
ggacccctgc taataaatct taccaatatt accgcatcac ttacggcgaa
120acaggaggca atagccctgt ccaggagttc actgtgcctc atgacctggt
tacagctacc 180atcagcggcc ttaaacctgg cgttgattat accatcactg
tgtatgctgt cactgaccat 240aaaccgcatg ctgacggtcc gcatacttac
catgaatacc caatttccat taattaccgc 300acagaaattg acaaaccatc
ccagcaccat caccaccacc ac 342428342DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 428atgggagttt
ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc caccagcctg 60ctgatcagct
gggacgctcc ggctgttact taccagtatt accgcatcac ttacggcgaa
120acaggaggca atagccctgt ccaggagttc actgtgcctc atgacctggt
tacagctacc 180atcagcggcc ttaaacctgg cgttgattat accatcactg
tgtatgctgt cactgactat 240aaaccgcatg ctgacggtcc gcatacttac
catgaatctc caatttccat taattaccgc 300acagaaattg acaaaccatc
ccagcaccat caccaccacc ac 342429342DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
429atgggagttt ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc
caccagcctg 60ctgatcagct ggacccctgc taataaatct taccaatatt accgcatcac
ttacggcgaa 120acaggaggca atagccctgt ccaggagttc actgtgcctc
atgacctggt tacagctacc 180atcagcggcc ttaaacctgg cgttgattat
accatcactg tgtatgctgt cactgaccat 240aaaccgcatg ctgacggtcc
gcatacttac catgaatctc caatttccat taattaccgc 300acagaaattg
acaaaccatc ccagcaccat caccaccacc ac 342430327DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
430atgggagttt ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc
caccagcctg 60ctgatcagct ggacccctgc taataaatct taccaatatt accgcatcac
ttacggcgaa 120acaggaggca atagccctgt ccaggagttc actgtgcctg
gtggtgttcg tacagctacc 180atcagcggcc ttaaacctgg cgttgattat
accatcactg tgtatgctgt cactaacatg 240atgcatgttg aatactctga
atacccaatt tccattaatt accgcacaga aattgacaaa 300ccatcccagc
accatcacca ccaccac 327431342DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 431atgggagttt
ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc caccagcctg 60ctgatcagct
gggacgctgg tgctgttact taccagtatt accgcatcac ttacggcgaa
120acaggaggca atagccctgt ccaggagttc actgtgcctc atgacctggt
tacagctacc 180atcagcggcc ttaaacctgg cgttgattat accatcactg
tgtatgctgt cactgaccat 240aaaccgcatg ctgacggtcc gcatacttac
catgaatacc caatttccat taattaccgc 300acagaaattg acaaaccatc
ccagcaccat caccaccacc ac 342432342DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 432atgggagttt
ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc caccagcctg 60ctgatcagct
gggacgctcc aacctcccgc taccaatatt accgcatcac ttacggcgaa
120acaggaggca atagccctgt ccaggagttc actgtgcctg gtggtctgtc
cacagctacc 180atcagcggcc ttaaacctgg cgttgattat accatcactg
tgtatgctgt cactgactat 240aaaccgcatg ctgacggtcc gcatacttac
catgaatctc caatttccat taattaccgc 300acagaaattg acaaaccatc
ccagcaccat caccaccacc ac 342433342DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 433atgggagttt
ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc caccagcctg 60ctgatcagct
gggacgctcc ggctgttact taccagtatt accgcatcac ttacggcgaa
120acaggaggca atagccctgt ccaggagttc actgtgcctt acgacgttta
cacagctacc 180atcagcggcc ttaaacctgg cgttgattat accatcactg
tgtatgctgt cactgactat 240aaaccgcacg ctgacggtcc gcacacttac
catgaatatc caatttccat taattaccgc 300acagaaattg acaaaccatc
ccagcaccat caccaccacc ac 342434327DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 434atgggagttt
ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc caccagcctg 60ctgatcagct
gggattcagg acgaggttcc tatcaatatt accgcatcac ttacggcgaa
120acaggaggca atagccctgt ccaggagttc actgtgcctt acgacgttta
cacagctacc 180atcagcggcc ttaaacctgg cgttgattat accatcactg
tgtatgctgt cactgacatg 240atgcatgttg aatacactga acatccaatt
tccattaatt accgcacaga aattgacaaa 300ccatcccagc accatcacca ccaccac
327435342DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 435atgggagttt ctgatgtgcc gcgcgacctg
gaagtggttg ctgccacccc caccagcctg 60ctgatcagct gggacgctcc ggctgttact
taccagtatt accgcatcac ttacggcgaa 120acaggaggca atagccctgt
ccaggagttc actgtgcctg gtggtgttcg tacagctacc 180atcagcggcc
ttaaacctgg cgttgattat accatcactg tgtatgctgt cactgactat
240aaaccgcatg ctgacggtcc gcatacttac catgaatctc caatttccat
taattaccgc 300acagaaattg acaaaccatc ccagcaccat caccaccacc ac
342436342DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 436atgggagttt ctgatgtgcc gcgcgacctg
gaagtggttg ctgccacccc caccagcctg 60ctgatcagct gggacgctcc aacctcccgc
taccaatatt accgcatcac ttacggcgaa 120acaggaggca atagccctgt
ccaggagttc actgtgcctc atgacctggt tacagctacc 180atcagcggcc
ttaaacctgg cgttgattat accatcactg tgtatgctgt cactgactat
240aaaccgcatg ctgacggtcc gcatacttac catgaatctc caatttccat
taattaccgc 300acagaaattg acaaaccatc ccagcaccat caccaccacc ac
342437342DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 437atgggagttt ctgatgtgcc gcgcgacctg
gaagtggttg ctgccacccc caccagcctg 60ctgatcagct gggacgctgg tgctgttact
taccagtatt accgcatcac ttacggcgaa 120acaggaggca atagccctgt
ccaggagttc actgtgcctg gtggtgttcg tacagctacc 180atcagcggcc
ttaaacctgg cgttgattat accatcactg tgtatgctgt cactgactat
240aaaccgcatg ctgacggtcc gcatacttac catgaatctc caatttccat
taattaccgc 300acagaaattg acaaaccatc ccagcaccat caccaccacc ac
342438327DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 438atgggagttt ctgatgtgcc gcgcgacctg
gaagtggttg ctgccacccc caccagcctg 60ctgatcagct gggacgctcc aacctcccgc
taccaatatt accgcatcac ttacggcgaa 120acaggaggca atagccctgt
ccaggagttc actgtgcctc atgacctggt tacagctacc 180atcagcggcc
ttaaacctgg cgttgattat accatcactg tgtatgctgt cactaacatg
240atgcatgttg aatactctga atacccaatt tccattaatt accgcacaga
aattgacaaa 300ccatcccagc accatcacca ccaccac 327439342DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
439atgggagttt ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc
caccagcctg 60ctgatcagct gggacgctgg tgctgttact taccagtatt accgcatcac
ttacggcgaa 120acaggaggca atagccctgt ccaggagttc actgtgcctg
gtggtgttcg tacagctacc 180atcagcggcc ttaaacctgg cgttgattat
accatcactg tgtatgctgt cactgactat 240aaaccgcacg ctgacggtcc
gcacacttac catgaatatc caatttccat taattaccgc 300acagaaattg
acaaaccatc ccagcaccat caccaccacc ac 342440327DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
440atgggagttt ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc
caccagcctg 60ctgatcagct ggtatcctgg ccaaccaaca tatcaatatt accgcatcac
ttacggcgaa 120acaggaggca atagccctgt ccaggagttc attgtgcctt
acctggttta cacagctacc 180atcagcggcc ttaaacctgg cgttgattat
accatcactg tgtatgctgt cactgactac 240gcttacaaag aatactctga
atacccaatt tccattaatt accgcacaga aattgacaaa 300ccatcccagc
accatcacca ccaccac 327441342DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 441atgggagttt
ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc caccagcctg 60ctgatcagct
ggcaaagttc aaccagccaa tatcaatatt accgcatcac ttacggcgaa
120acaggaggca atagccctgt ccaggagttc actgtgcctg gtggtgttcg
tacagctacc 180atcagcggcc ttaaacctgg cgttgattat accatcactg
tgtatgctgt cactgaccat 240aaaccgcatg ctgacggtcc gcatacttac
catgaatctc caatttccat taattaccgc 300acagaaattg acaaaccatc
ccagcaccat caccaccacc ac 342442669DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 442atgggagttt
ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc caccagcctg 60ctgatcagct
ggttgccggg caagctgagg taccaatatt accgcatcac ttacggcgaa
120acaggaggca atagccctgt ccaggagttc actgtgcctc atgacctgcg
tacagctacc 180atcagcggcc ttaaacctgg cgttgattat accatcactg
tgtatgctgt cactaacatg 240atgcatgttg aatactctga atacccaatt
tccattaatt accgcacaga aattgacaaa 300ggtagcggct ctggttccgg
cagcggctcc ggcagcggct ctggcagcgg ttctggttcc 360gtttctgatg
tgccgcgcga cctggaagtg gttgctgcca cccccaccag cctgctgatc
420agctggtctg cgcgtctgaa agttgcgcga tattaccgca tcacttacgg
cgaaacagga 480ggcaatagcc ctgtccagga gttcactgtg cctaaaaacg
tttacacagc taccatcagc 540ggccttaaac ctggcgttga ttataccatc
actgtgtatg ctgtcactag gttccgcgac 600taccagccaa tttccattaa
ttaccgcaca gaaattgaca aaccatgcca gcaccaccac 660caccaccac
669443669DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 443atgggagttt ctgatgtgcc gcgcgacctg
gaagtggttg ctgccacccc caccagcctg 60ctgatcagct ggtctgcgcg tctgaaagtt
gcgcgatatt accgcatcac ttacggcgaa 120acaggaggca atagccctgt
ccaggagttc actgtgccta aaaacgttta cacagctacc 180atcagcggcc
ttaaacctgg cgttgattat accatcactg tgtatgctgt cactaggttc
240cgcgactacc agccaatttc cattaattac cgcacagaaa ttgacaaagg
tagcggctct 300ggttccggca gcggctccgg cagcggctct ggcagcggtt
ctggttccgt ttctgatgtg 360ccgcgcgacc tggaagtggt tgctgccacc
cccaccagcc tgctgatcag ctggttgccg 420ggcaagctga ggtaccaata
ttaccgcatc acttacggcg aaacaggagg caatagccct 480gtccaggagt
tcactgtgcc tcatgacctg cgtacagcta ccatcagcgg ccttaaacct
540ggcgttgatt ataccatcac tgtgtatgct gtcactaaca tgatgcatgt
tgaatactct 600gaatacccaa tttccattaa ttaccgcaca gaaattgaca
aaccatgcca gcaccaccac 660caccaccac 669444669DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
444atgggagttt ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc
caccagcctg 60ctgatcagct gggtggccgg ggcggaggac taccaatatt accgcatcac
ttacggcgaa 120acaggaggca atagccctgt ccaggagttc actgtgcctc
atgacctggt tacagctacc 180atcagcggcc ttaaacctgg cgttgattat
accatcactg tgtatgctgt cactgacatg 240atgcatgttg aatacactga
acatccaatt tccattaatt accgcacaga aattgacaaa 300ggtagcggct
ctggttccgg cagcggctcc ggcagcggct ctggcagcgg ttctggttcc
360gtttctgatg tgccgcgcga cctggaagtg gttgctgcca cccccaccag
cctgctgatc 420agctggtctg cgcgtctgaa agttgcgcga tattaccgca
tcacttacgg cgaaacagga 480ggcaatagcc ctgtccagga gttcactgtg
cctaaaaacg tttacacagc taccatcagc 540ggccttaaac ctggcgttga
ttataccatc actgtgtatg ctgtcactag gttccgcgac 600taccagccaa
tttccattaa ttaccgcaca gaaattgaca aaccatgcca gcaccaccac 660caccaccac
669445669DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 445atgggagttt ctgatgtgcc gcgcgacctg
gaagtggttg ctgccacccc caccagcctg 60ctgatcagct ggtctgcgcg tctgaaagtt
gcgcgatatt accgcatcac ttacggcgaa 120acaggaggca atagccctgt
ccaggagttc actgtgccta aaaacgttta cacagctacc 180atcagcggcc
ttaaacctgg cgttgattat accatcactg tgtatgctgt cactaggttc
240cgcgactacc agccaatttc cattaattac cgcacagaaa ttgacaaagg
tagcggctct 300ggttccggca gcggctccgg cagcggctct ggcagcggtt
ctggttccgt ttctgatgtg 360ccgcgcgacc tggaagtggt tgctgccacc
cccaccagcc tgctgatcag ctgggtggcc 420ggggcggagg actaccaata
ttaccgcatc acttacggcg aaacaggagg caatagccct 480gtccaggagt
tcactgtgcc tcatgacctg gttacagcta ccatcagcgg ccttaaacct
540ggcgttgatt ataccatcac tgtgtatgct gtcactgaca tgatgcatgt
tgaatacact 600gaacatccaa tttccattaa ttaccgcaca gaaattgaca
aaccatgcca gcaccaccac 660caccaccac 669446684DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
446atgggagttt ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc
caccagcctg 60ctgatcagct gggattcagg acgaggttcc tatcaatatt accgcatcac
ttacggcgaa 120acaggaggca atagccctgt ccaggagttc actgtgcctg
gtccggttca tacagctacc 180atcagcggcc ttaaacctgg cgttgattat
accatcactg tgtatgctgt cactgaccat 240aaaccgcatg ctgacggtcc
gcatacttac catgaatctc caatttccat taattaccgc 300acagaaattg
acaaaggtag cggctctggt tccggcagcg gctccggcag cggctctggc
360agcggttctg gttccgtttc tgatgtgccg cgcgacctgg aagtggttgc
tgccaccccc 420accagcctgc tgatcagctg gtctgcgcgt ctgaaagttg
cgcgatatta ccgcatcact 480tacggcgaaa caggaggcaa tagccctgtc
caggagttca ctgtgcctaa aaacgtttac 540acagctacca tcagcggcct
taaacctggc gttgattata ccatcactgt gtatgctgtc 600actaggttcc
gcgactacca gccaatttcc attaattacc gcacagaaat tgacaaacca
660tgccagcacc accaccacca ccac 684447684DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
447atgggagttt ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc
caccagcctg 60ctgatcagct ggtctgcgcg tctgaaagtt gcgcgatatt accgcatcac
ttacggcgaa 120acaggaggca atagccctgt ccaggagttc actgtgccta
aaaacgttta cacagctacc 180atcagcggcc ttaaacctgg cgttgattat
accatcactg tgtatgctgt cactaggttc 240cgcgactacc agccaatttc
cattaattac cgcacagaaa ttgacaaagg tagcggctct 300ggttccggca
gcggctccgg cagcggctct ggcagcggtt ctggttccgt ttctgatgtg
360ccgcgcgacc tggaagtggt tgctgccacc cccaccagcc tgctgatcag
ctgggattca 420ggacgaggtt cctatcaata ttaccgcatc acttacggcg
aaacaggagg caatagccct 480gtccaggagt tcactgtgcc tggtccggtt
catacagcta ccatcagcgg ccttaaacct 540ggcgttgatt ataccatcac
tgtgtatgct gtcactgacc ataaaccgca tgctgacggt 600ccgcatactt
accatgaatc tccaatttcc attaattacc gcacagaaat tgacaaacca
660tgccagcacc accaccacca ccac 684448684DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
448atgggtgttt ctgatgttcc gcgtgatctg gaggttgttg cagcaacccc
gaccagcctg 60ctgatttctt ggtgggcacc ggttgatcgt tatcagtatt atcgcatcac
ctatggtgaa 120accggtggta attctccggt tcaggaattt accgttcctc
gcgacgttta taccgcaacc 180attagcggtc tgaaaccggg tgttgattac
accattaccg tttacgccgt taccgattat 240aaaccgcatg cagatggtcc
gcatacctat catgaaagcc cgattagcat taactatcgc 300accgaaattg
ataaaggtag cggtagcggt tcaggtagcg gatcaggttc tggttctggt
360agtggtagcg gcagcgtttc agatgtgcct cgcgacctgg aagtggtggc
agccacaccg 420acttctctgc tgattagctg gtctgcacgt ctgaaagttg
cccgttatta ccgtattact 480tatggcgaaa caggcggaaa tagccctgtg
caagaattta ccgtgccgaa aaatgtgtac 540acagccacca tctctggcct
gaaacctggc gtggactaca caatcacagt ttatgcagtg 600acccgttttc
gtgattatca gccgatcagc atcaattatc gtacagagat cgataaaccg
660tgccagcatc accaccatca tcac 684449684DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
449atgggtgttt ctgatgttcc gcgtgatctg gaggttgttg cagcaacccc
gaccagcctg 60ctgatttctt ggacccaggg tagcacacat tatcagtatt atcgcatcac
ctatggtgaa 120accggtggta attctccggt tcaggaattt accgttcctg
gtatggttta taccgcaacc 180attagcggtc tgaaaccggg tgttgattac
accattaccg tttacgccgt taccgattat 240ttcgatcggt ccacccatga
atataaatat cggaccaccc cgattagcat taactatcgc 300accgaaattg
ataaaggtag cggtagcggt tcaggtagcg gatcaggttc tggttctggt
360agtggtagcg gcagcgtttc agatgtgcct cgcgacctgg aagtggtggc
agccacaccg 420acttctctgc tgattagctg gtctgcacgt ctgaaagttg
cccgttatta ccgtattact 480tatggcgaaa caggcggaaa tagccctgtg
caagaattta ccgtgccgaa aaatgtgtac 540acagccacca tctctggcct
gaaacctggc gtggactaca caatcacagt ttatgcagtg 600acccgttttc
gtgattatca gccgatcagc atcaattatc gtacagagat cgataaaccg
660tgccagcatc accaccatca tcac 684450684DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
450atgggtgttt ctgatgttcc gcgtgatctg gaggttgttg cagcaacccc
gaccagcctg 60ctgatttctt ggcatgaacg tgatggtagc cgtcagtatt atcgcatcac
ctatggtgaa 120accggtggta attctccggt tcaggaattt accgttcctg
gcggtgttcg taccgcaacc 180attagcggtc tgaaaccggg tgttgattac
accattaccg tttacgccgt taccgattat 240ttcaatccga ccacccatga
atatatttat cagaccaccc cgattagcat taactatcgc 300accgaaattg
ataaaggtag cggtagcggt tcaggtagcg gatcaggttc tggttctggt
360agtggtagcg gcagcgtttc agatgtgcct cgcgacctgg aagtggtggc
agccacaccg 420acttctctgc tgattagctg gtctgcacgt ctgaaagttg
cccgttatta ccgtattact 480tatggcgaaa caggcggaaa tagccctgtg
caagaattta ccgtgccgaa aaatgtgtac 540acagccacca tctctggcct
gaaacctggc gtggactaca caatcacagt ttatgcagtg 600acccgttttc
gtgattatca gccgatcagc atcaattatc gtacagagat cgataaaccg
660tgccagcatc accaccatca tcac 684451684DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
451atgggtgttt ctgatgttcc gcgtgatctg gaggttgttg cagcaacccc
gaccagcctg 60ctgatttctt ggtattggga aggtctgccg tatcagtatt atcgcatcac
ctatggtgaa 120accggtggta attctccggt tcaggaattc accgttcctc
gcgacgttaa taccgcaacc 180attagcggtc tgaaaccggg tgttgattac
accattaccg tttacgccgt taccgattgg 240tacaaccctg atacccatga
atatatttat cataccattc cgattagcat taactatcgc 300accgaaattg
ataaaggtag cggtagcggt tcaggtagcg gatcaggttc tggttctggt
360agtggtagcg gcagcgtttc agatgtgcct cgcgacctgg aagtggtggc
agccacaccg 420acttctctgc tgattagctg gtctgcacgt ctgaaagttg
cccgttatta ccgtattact 480tatggcgaaa caggcggaaa tagccctgtg
caagaattta ccgtgccgaa aaatgtgtac 540acagccacca tctctggcct
gaaacctggc gtggactaca caatcacagt ttatgcagtg 600acccgttttc
gtgattatca gccgatcagc atcaattatc gtacagagat cgataaaccg
660tgccagcatc accaccatca tcac 684452684DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
452atgggtgttt ctgatgttcc gcgtgatctg gaggttgttg cagcaacccc
gaccagcctg 60ctgatttctt gggcaagcaa tcgtggcacc tatcagtatt atcgcatcac
ctatggtgaa 120accggtggta attctccggt tcaggaattt accgttcctg
gcggtgtttc taccgcaacc 180attagcggtc tgaaaccggg tgttgattac
accattaccg tttacgccgt taccgatgca 240tttaatccga ccacccatga
atataattat tttaccaccc cgattagcat taactatcgc 300accgaaattg
ataaaggtag cggtagcggt tcaggtagcg gatcaggttc tggttctggt
360agtggtagcg gcagcgtttc agatgtgcct cgcgacctgg aagtggtggc
agccacaccg 420acttctctgc tgattagctg gtctgcacgt ctgaaagttg
cccgttatta ccgtattact 480tatggcgaaa caggcggaaa tagccctgtg
caagaattta ccgtgccgaa aaatgtgtac 540acagccacca tctctggcct
gaaacctggc gtggactaca caatcacagt ttatgcagtg 600acccgttttc
gtgattatca gccgatcagc atcaattatc gtacagagat cgataaaccg
660tgccagcatc accaccatca tcac 684453684DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
453atgggtgttt ctgatgttcc gcgtgatctg gaggttgttg cagcaacccc
gaccagcctg 60ctgatttctt gggatgcacc gacctctcgt tatcagtatt atcgcatcac
ctatggtgaa 120accggtggta attctccggt tcaggaattt accgttcctg
gcggtctgag caccgcaacc 180attagcggtc tgaaaccggg tgttgattac
accattaccg tttacgccgt taccgattat
240aaaccgcatg cagatggtcc gcatacctat catgaaagcc cgattagcat
taactatcgc 300accgaaattg ataaaggtag cggtagcggt tcaggtagcg
gatcaggttc tggttctggt 360agtggtagcg gcagcgtttc agatgtgcct
cgcgacctgg aagtggtggc agccacaccg 420acttctctgc tgattagctg
gtctgcacgt ctgaaagttg cccgttatta ccgtattact 480tatggcgaaa
caggcggaaa tagccctgtg caagaattta ccgtgccgaa aaatgtgtac
540acagccacca tctctggcct gaaacctggc gtggactaca caatcacagt
ttatgcagtg 600acccgttttc gtgattatca gccgatcagc atcaattatc
gtacagagat cgataaaccg 660tgccagcatc accaccatca tcac
684454684DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 454atgggtgttt ctgatgttcc gcgtgatctg
gaggttgttg cagcaacccc gaccagcctg 60ctgatttctt gggatgcagg tgcagttacc
tatcagtatt atcgcatcac ctatggtgaa 120accggtggta attctccggt
tcaggaattt accgttcctg gcggtgttcg taccgcaacc 180attagcggtc
tgaaaccggg tgttgattac accattaccg tttacgccgt taccgattat
240aaaccgcatg cagatggtcc gcatacctat catgaatatc cgattagcat
taactatcgc 300accgaaattg ataaaggtag cggtagcggt tcaggtagcg
gatcaggttc tggttctggt 360agtggtagcg gcagcgtttc agatgtgcct
cgcgacctgg aagtggtggc agccacaccg 420acttctctgc tgattagctg
gtctgcacgt ctgaaagttg cccgttatta ccgtattact 480tatggcgaaa
caggcggaaa tagccctgtg caagaattta ccgtgccgaa aaatgtgtac
540acagccacca tctctggcct gaaacctggc gtggactaca caatcacagt
ttatgcagtg 600acccgttttc gtgattatca gccgatcagc atcaattatc
gtacagagat cgataaaccg 660tgccagcatc accaccatca tcac
684455684DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 455atgggtgttt ctgatgttcc gcgtgatctg
gaagttgttg cagcaacccc gaccagcctg 60ctgattagct ggtctgcacg tctgaaagtt
gcccgttatt atcgcattac ctatggtgaa 120accggtggta attctccggt
tcaggaattt accgttccga aaaatgttta taccgcaacc 180attagcggtc
tgaaaccggg tgttgattac accattaccg tttatgcagt tacccgtttt
240cgtgattatc agccgattag cattaactat cgcaccgaaa ttgataaagg
tagcggtagc 300ggttctggta gcggttcagg ttctggttct ggtagtggta
gcggcagcgt ttcagacgtg 360cctcgtgatt tagaagtggt ggcagccaca
ccgacctcac tgctgatttc ttggtgggca 420ccggttgatc gttatcagta
ttatcgcatc acatacggcg aaacaggcgg aaatagccct 480gtgcaagaat
tcaccgtacc gcgtgatgtg tataccgcca caatttctgg tttaaaacct
540ggcgtggact acacaatcac agtttatgcc gtgaccgatt ataaaccgca
tgcagatggt 600ccgcatacct atcatgaaag cccgatctct atcaattatc
gcacagagat cgataaaccg 660tgtcagcatc accaccatca tcac
684456684DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 456atgggtgttt ctgatgttcc gcgtgatctg
gaagttgttg cagcaacccc gaccagcctg 60ctgattagct ggtctgcacg tctgaaagtt
gcccgttatt atcgcattac ctatggtgaa 120accggtggta attctccggt
tcaggaattt accgttccga aaaatgttta taccgcaacc 180attagcggtc
tgaaaccggg tgttgattac accattaccg tttatgcagt tacccgtttt
240cgtgattatc agccgattag cattaactat cgcaccgaaa ttgataaagg
tagcggtagc 300ggttctggta gcggttcagg ttctggttct ggtagtggta
gcggcagcgt ttcagacgtg 360cctcgtgatt tagaagtggt ggcagccaca
ccgacctcac tgctgatttc ttggacccag 420ggtagcacac attatcagta
ttatcgcatc acatacggcg aaacaggcgg aaatagccct 480gtgcaagaat
tcaccgtacc gggtatggtg tataccgcca caatttctgg tttaaaacct
540ggcgtggact acacaatcac agtttatgcc gtgaccgatt atttcgatcg
cagcacccat 600gaatataaat atcgtaccac cccgatctct atcaattatc
gcacagagat cgataaaccg 660tgtcagcatc accaccatca tcac
684457684DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 457atgggtgttt ctgatgttcc gcgtgatctg
gaagttgttg cagcaacccc gaccagcctg 60ctgattagct ggtctgcacg tctgaaagtt
gcccgttatt atcgcattac ctatggtgaa 120accggtggta attctccggt
tcaggaattt accgttccga aaaatgttta taccgcaacc 180attagcggtc
tgaaaccggg tgttgattac accattaccg tttatgcagt tacccgtttt
240cgtgattatc agccgattag cattaactat cgcaccgaaa ttgataaagg
tagcggtagc 300ggttctggta gcggttcagg ttctggttct ggtagtggta
gcggcagcgt ttcagacgtg 360cctcgtgatt tagaagtggt ggcagccaca
ccgacctcac tgctgatttc ttggcatgaa 420cgtgatggta gccgtcagta
ttatcgcatc acatacggcg aaacaggcgg aaatagccct 480gtgcaagaat
tcaccgtacc gggtggtgtt cgtaccgcca caatttctgg tttaaaacct
540ggcgtggact acacaatcac agtttatgcc gtgaccgatt atttcaatcc
gaccacccac 600gaatatattt atcagaccac cccgatctct atcaattatc
gcacagagat cgataaaccg 660tgtcagcatc accaccatca tcac
684458684DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 458atgggtgttt ctgatgttcc gcgtgatctg
gaagttgttg cagcaacccc gaccagcctg 60ctgattagct ggtctgcacg tctgaaagtt
gcccgttatt atcgcattac ctatggtgaa 120accggtggta attctccggt
tcaggaattt accgttccga aaaatgttta taccgcaacc 180attagcggtc
tgaaaccggg tgttgattac accattaccg tttatgcagt tacccgtttt
240cgtgattatc agccgattag cattaactat cgcaccgaaa ttgataaagg
tagcggtagc 300ggttctggta gcggttcagg ttctggttct ggtagtggta
gcggcagcgt ttcagacgtg 360cctcgtgatt tagaagtggt ggcagccaca
ccgacctcac tgctgatttc ttgggatgca 420ccgaccagcc gttatcagta
ttatcgcatc acatacggcg aaacaggcgg aaatagccct 480gtgcaagaat
tcaccgtacc gggtggtctg agcaccgcca caatttctgg tttaaaacct
540ggcgtggact acacaatcac agtttatgcc gtgaccgatt ataaaccgca
tgcagatggt 600ccgcatacct atcatgaaag cccgatctct atcaattatc
gcacagagat cgataaaccg 660tgtcagcatc accaccatca tcac
684459684DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 459atgggtgttt ctgatgttcc gcgtgatctg
gaagttgttg cagcaacccc gaccagcctg 60ctgattagct ggtctgcacg tctgaaagtt
gcccgttatt atcgcattac ctatggtgaa 120accggtggta attctccggt
tcaggaattt accgttccga aaaatgttta taccgcaacc 180attagcggtc
tgaaaccggg tgttgattac accattaccg tttatgcagt tacccgtttt
240cgtgattatc agccgattag cattaactat cgcaccgaaa ttgataaagg
tagcggtagc 300ggttctggta gcggttcagg ttctggttct ggtagtggta
gcggcagcgt ttcagacgtg 360cctcgtgatt tagaagtggt ggcagccaca
ccgacctcac tgctgatttc ttgggatgcc 420ggtgcagtta cctatcagta
ttatcgcatc acatacggcg aaacaggcgg aaatagccct 480gtgcaagaat
tcaccgtacc gggtggtgtt cgtaccgcca caatttctgg tttaaaacct
540ggcgtggact acacaatcac agtttatgcc gtgaccgatt ataaaccgca
tgcagatggt 600ccgcatacct atcatgaata tccgatctct atcaattatc
gcacagagat cgataaaccg 660tgtcagcatc accaccatca tcac
684460666DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 460atgggcgtga gtgatgttcc gcgtgatctg
gaagtggttg cagcaacccc gacgagcctg 60ctgattagct ggtctgcccg cctgaaagtg
gcacgttatt accgcatcac ctacggcgaa 120acgggcggta actctccggt
tcaggaattt accgtgccga aaaatgttta taccgcaacg 180attagcggcc
tgaaaccggg tgtggattat accatcacgg tgtacgcggt tacccgtttc
240cgcgattacc agccgattag catcaactat cgtacggaaa ttgaaaaagg
ctctggttgc 300ggcagtggta gcggctctgg tagtggcagc ggttctggca
gtggtagcgt gtctgacgtc 360ccgcgcgacc tggaagttgt tgcagcgacc
ccgaccagcc tgctgattag ttggtgggcc 420ccggtggatc gttaccagta
ttaccgcatc acctatggcg aaaccggtgg taacagcccg 480gtgcaagaat
ttaccgtgcc gcgtgatgtt tataccgcga ccatctctgg tctgaaaccg
540ggcgttgact acacgattac cgtttacgcg gttaccgatt ataaaccgca
tgccgatggt 600ccgcatacgt accacgaaag tccgattagc atcaattatc
ggaccgaaca tcaccatcac 660catcac 666461678DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
461atgggcgtgt ctgatgttcc gcgtgatctg gaagtggttg cggccacccc
gacgagtctg 60ctgattagct ggtctgcccg cctgaaagtg gcacgttatt accgcatcac
ctacggcgaa 120acgggcggta acagcccggt tcaggaattt accgtgccga
aaaatgttta taccgcaacg 180atttctggcc tgaaaccggg tgtggattat
accatcacgg tgtacgcggt tacccgtttc 240cgcgattacc agccgattag
catcaactat cgtacggaaa ttgaaaaagg cagtggtagc 300ggctctggta
gtggcagcgg ttctggcagt ggtagcggct ctggtagtgt aagcgacgtc
360ccgcgcgatc tggaagtggt tgcagcgacc ccgacgagcc tgctgatttc
ttggtgggcc 420ccggtggatc gttaccagta ttaccgcatc acctatggcg
aaaccggtgg caattctccg 480gtgcaagaat tcaccgtgcc gcgtgatgtt
tataccgcga cgattagcgg tctgaaaccg 540ggcgttgact acacgattac
cgtgtacgcg gttaccgatt ataaaccgca tgccgatggt 600ccgcatacgt
accacgaatc tccgattagt atcaattatc ggaccgaagg cagtggttgc
660catcaccatc accatcac 678462666DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 462atgggcgtga
gtgatgttcc gcgtgatctg gaagtggttg cagcaacccc gacgagcctg 60ctgattagct
ggtctgcccg cctgaaagtg gcacgttatt accgcatcac ctacggcgaa
120acgggcggta acagcccggt tcaggaattt accgtgccga aaaatgttta
taccgcaacg 180atttgcggcc tgaaaccggg tgtggattat accatcacgg
tgtacgcggt tacccgtttc 240cgcgattacc agccgattag catcaactat
cgtacggaaa ttgaaaaagg cagtggtagc 300ggctctggta gtggcagcgg
ttctggcagt ggtagcggct ctggtagtgt aagcgacgtc 360ccgcgcgacc
tggaagttgt tgcagcgacg ccgacgagcc tgctgatctc ttggtgggcc
420ccggtggatc gttaccagta ttaccgcatc acctatggcg aaaccggtgg
taactctccg 480gtgcaagaat ttaccgtgcc gcgtgatgtt tataccgcga
cgatttctgg tctgaaaccg 540ggcgttgact acacgattac cgtttacgcg
gttaccgatt ataaaccgca tgccgatggt 600ccgcatacgt accacgaatc
tccgattagt atcaattatc ggaccgaaca tcaccatcac 660catcac
666463666DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 463atgggcgtga gtgatgttcc gcgtgatctg
gaagtggttg cagcaacccc gacgagcctg 60ctgattagct ggtctgcccg cctgaaagtg
gcacgttatt accgcatcac ctacggcgaa 120acgggcggta actctccggt
tcaggaattt accgtgccga aaaatgttta taccgcaacg 180attagcggcc
tgaaaccggg tgtggattat accatcacgg tgtacgcggt tacccgtttc
240cgcgattacc agccgattag catcaactat cgtacggaaa ttgaaaaagg
cagtggtagc 300ggctctggta gtggcagcgg ttctggcagt ggtagcggct
ctggtagtgt aagcgacgtc 360ccgcgcgacc tggaagttgt tgcagcgacg
ccgacgagcc tgctgatctc ttggtgggcc 420ccggtggatc gttaccagta
ttaccgcatc acctatggcg aaaccggtgg taacagcccg 480gtgcaagaat
ttaccgtgcc gcgtgatgtt tataccgcga cgatttgtgg tctgaaaccg
540ggcgttgact acacgattac cgtttacgcg gttaccgatt ataaaccgca
tgccgatggt 600ccgcatacgt accacgaatc tccgattagt atcaattatc
ggaccgaaca tcaccatcac 660catcac 666464666DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
464atgggcgtga gtgatgttcc gcgtgatctg gaagtggttg cagcaacccc
gacgagcctg 60ctgattagct ggtctgcccg cctgaaagtg gcacgttatt accgcatcac
ctacggcgaa 120acgggcggta actctccggt tcaggaattt accgtgccga
aaaatgttta taccgcaacg 180attagcggcc tgaaaccggg tgtggattat
accatcacgg tgtacgcggt tacccgtttc 240cgcgattacc agccgatttg
catcaactat cgtacggaaa ttgaaaaagg cagtggtagc 300ggctctggta
gtggcagcgg ttctggcagt ggtagcggct ctggtagtgt aagcgacgtc
360ccgcgcgacc tggaagttgt tgcagcgacg ccgacgagcc tgctgatctc
ttggtgggcc 420ccggtggatc gttaccagta ttaccgcatc acctatggcg
aaaccggtgg taacagcccg 480gtgcaagaat ttaccgtgcc gcgtgatgtt
tataccgcga ccatctctgg tctgaaaccg 540ggcgttgact acacgattac
cgtttacgcg gttaccgatt ataaaccgca tgccgatggt 600ccgcatacgt
accacgaatc tccgattagt atcaattatc ggaccgaaca tcaccatcac 660catcac
666465666DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 465atgggcgtga gcgatgttcc gcgtgatctg
gaagtggttg cagcaacccc gaccagcctg 60ctgattagct ggtctgcccg cctgaaagtg
gcacgttatt accgcatcac ctacggcgaa 120acgggcggta acagtccggt
tcaggaattt accgtgccga aaaatgttta taccgcaacg 180attagcggcc
tgaaaccggg tgtggattat accatcacgg tgtacgcggt tacccgtttc
240cgcgattacc agccgattag catcaactat cgtacggaaa ttgaaaaagg
cagtggtagc 300ggctctggta gtggcagcgg ttctggcagt ggtagcggct
ctggtagtgt aagcgacgtc 360ccgcgcgacc tggaagttgt tgcagcgacg
ccgaccagcc tgctgatcag ttggtgggcc 420ccggtggatc gttaccagta
ttaccgcatc acctatggcg aaaccggtgg taacagcccg 480gtgcaagaat
tcaccgtgcc gcgtgatgtt tataccgcga ccatctctgg tctgaaaccg
540ggcgttgact acacgattac cgtttacgcg gttaccgatt ataaaccgca
tgccgatggt 600ccgcatacgt accacgaaag cccgatttgc atcaattatc
ggaccgaaca tcaccatcac 660catcac 666466190PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
466Glu Val Val Ala Ala Thr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1
5 10 15Xaa Xaa Xaa Xaa Xaa Ser Leu Leu Ile Xaa Xaa Xaa Xaa Xaa Xaa
Xaa 20 25 30Xaa Xaa Xaa Ser Trp His Glu Arg Asp Gly Ser Arg Gln Xaa
Xaa Xaa 35 40 45Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr Tyr Arg Ile Thr Tyr
Gly Glu Xaa 50 55 60Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa65 70 75 80Xaa Xaa Xaa Gln Glu Phe Thr Val Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95Xaa Xaa Pro Gly Gly Val Arg Thr Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 100 105 110Xaa Xaa Ala Thr Ile Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 115 120 125Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Asp Tyr Thr Ile Thr Val Tyr 130 135 140Ala Val Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Thr Asp Tyr Phe145 150 155
160Asn Pro Thr Thr His Glu Tyr Ile Tyr Gln Thr Thr Pro Xaa Xaa Xaa
165 170 175Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ile Ser Ile Asn Tyr Arg Thr
180 185 190467190PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 467Glu Val Val Ala Ala Thr Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10 15Xaa Xaa Xaa Xaa Xaa Ser Leu
Leu Ile Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30Xaa Xaa Xaa Ser Trp Trp
Ala Pro Val Asp Arg Tyr Gln Xaa Xaa Xaa 35 40 45Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Tyr Tyr Arg Ile Thr Tyr Gly Glu Xaa 50 55 60Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa65 70 75 80Xaa Xaa
Xaa Gln Glu Phe Thr Val Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95Xaa
Xaa Pro Arg Asp Val Tyr Thr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 100 105
110Xaa Xaa Ala Thr Ile Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
115 120 125Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asp Tyr Thr Ile Thr
Val Tyr 130 135 140Ala Val Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Thr Asp Tyr Lys145 150 155 160Pro His Ala Asp Gly Pro His Thr Tyr
His Glu Ser Pro Xaa Xaa Xaa 165 170 175Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Ile Ser Ile Asn Tyr Arg Thr 180 185 190468190PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
468Glu Val Val Ala Ala Thr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1
5 10 15Xaa Xaa Xaa Xaa Xaa Ser Leu Leu Ile Xaa Xaa Xaa Xaa Xaa Xaa
Xaa 20 25 30Xaa Xaa Xaa Ser Trp Thr Gln Gly Ser Thr His Tyr Gln Xaa
Xaa Xaa 35 40 45Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr Tyr Arg Ile Thr Tyr
Gly Glu Xaa 50 55 60Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa65 70 75 80Xaa Xaa Xaa Gln Glu Phe Thr Val Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95Xaa Xaa Pro Gly Met Val Tyr Thr Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 100 105 110Xaa Xaa Ala Thr Ile Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 115 120 125Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Asp Tyr Thr Ile Thr Val Tyr 130 135 140Ala Val Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Thr Asp Tyr Phe145 150 155
160Asp Arg Ser Thr His Glu Tyr Lys Tyr Arg Thr Thr Pro Xaa Xaa Xaa
165 170 175Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ile Ser Ile Asn Tyr Arg Thr
180 185 190469190PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 469Glu Val Val Ala Ala Thr Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10 15Xaa Xaa Xaa Xaa Xaa Ser Leu
Leu Ile Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30Xaa Xaa Xaa Ser Trp Tyr
Trp Glu Gly Leu Pro Tyr Gln Xaa Xaa Xaa 35 40 45Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Tyr Tyr Arg Ile Thr Tyr Gly Glu Xaa 50 55 60Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa65 70 75 80Xaa Xaa
Xaa Gln Glu Phe Thr Val Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95Xaa
Xaa Pro Arg Asp Val Asn Thr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 100 105
110Xaa Xaa Ala Thr Ile Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
115 120 125Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asp Tyr Thr Ile Thr
Val Tyr 130 135 140Ala Val Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Thr Asp Trp Tyr145 150 155 160Asn Pro Asp Thr His Glu Tyr Ile Tyr
His Thr Ile Pro Xaa Xaa Xaa 165 170
175Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ile Ser Ile Asn Tyr Arg Thr 180 185
190470190PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 470Glu Val Val Ala Ala Thr Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa1 5 10 15Xaa Xaa Xaa Xaa Xaa Ser Leu Leu Ile
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30Xaa Xaa Xaa Ser Trp Ala Ser Asn
Arg Gly Thr Tyr Gln Xaa Xaa Xaa 35 40 45Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Tyr Tyr Arg Ile Thr Tyr Gly Glu Xaa 50 55 60Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa65 70 75 80Xaa Xaa Xaa Gln
Glu Phe Thr Val Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95Xaa Xaa Pro
Gly Gly Val Ser Thr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 100 105 110Xaa
Xaa Ala Thr Ile Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 115 120
125Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asp Tyr Thr Ile Thr Val Tyr
130 135 140Ala Val Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Thr Asp
Ala Phe145 150 155 160Asn Pro Thr Thr His Glu Tyr Asn Tyr Phe Thr
Thr Pro Xaa Xaa Xaa 165 170 175Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ile Ser
Ile Asn Tyr Arg Thr 180 185 190471190PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
471Glu Val Val Ala Ala Thr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1
5 10 15Xaa Xaa Xaa Xaa Xaa Ser Leu Leu Ile Xaa Xaa Xaa Xaa Xaa Xaa
Xaa 20 25 30Xaa Xaa Xaa Ser Trp Asp Ala Pro Thr Ser Arg Tyr Gln Xaa
Xaa Xaa 35 40 45Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr Tyr Arg Ile Thr Tyr
Gly Glu Xaa 50 55 60Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa65 70 75 80Xaa Xaa Xaa Gln Glu Phe Thr Val Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95Xaa Xaa Pro Gly Gly Leu Ser Thr Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 100 105 110Xaa Xaa Ala Thr Ile Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 115 120 125Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Asp Tyr Thr Ile Thr Val Tyr 130 135 140Ala Val Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Thr Asp Tyr Lys145 150 155
160Pro His Ala Asp Gly Pro His Thr Tyr His Glu Ser Pro Xaa Xaa Xaa
165 170 175Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ile Ser Ile Asn Tyr Arg Thr
180 185 190472190PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 472Glu Val Val Ala Ala Thr Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10 15Xaa Xaa Xaa Xaa Xaa Ser Leu
Leu Ile Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30Xaa Xaa Xaa Ser Trp Asp
Ala Gly Ala Val Thr Tyr Gln Xaa Xaa Xaa 35 40 45Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Tyr Tyr Arg Ile Thr Tyr Gly Glu Xaa 50 55 60Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa65 70 75 80Xaa Xaa
Xaa Gln Glu Phe Thr Val Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95Xaa
Xaa Pro Gly Gly Val Arg Thr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 100 105
110Xaa Xaa Ala Thr Ile Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
115 120 125Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asp Tyr Thr Ile Thr
Val Tyr 130 135 140Ala Val Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Thr Asp Tyr Lys145 150 155 160Pro His Ala Asp Gly Pro His Thr Tyr
His Glu Tyr Pro Xaa Xaa Xaa 165 170 175Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Ile Ser Ile Asn Tyr Arg Thr 180 185 19047310PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 473Asp
Tyr Xaa Gly Lys Pro Tyr Xaa Glu Tyr1 5 1047410PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 474Asp
Tyr Xaa Tyr Xaa Pro Tyr Xaa Glu Tyr1 5 1047510PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 475Xaa
Tyr Xaa Xaa Xaa Glu Tyr Xaa Glu Xaa1 5 1047610PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 476Asp
Tyr Tyr Xaa Xaa Xaa Xaa Xaa Tyr Xaa1 5 1047710PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 477Asp
Tyr Tyr Xaa Xaa Xaa Thr Xaa Tyr Xaa1 5 1047810PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 478Xaa
Met Met His Val Xaa Tyr Xaa Glu Tyr1 5 1047910PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 479Asp
Tyr Met His Xaa Xaa Tyr Xaa Glu Tyr1 5 1048010PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 480Asp
Xaa Tyr His Xaa Xaa Xaa Xaa Tyr Gly1 5 1048115PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 481Asp
Xaa Xaa Asn Pro Xaa Thr His Glu Tyr Xaa Tyr Xaa Xaa Xaa1 5 10
1548215PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 482Asp Xaa Xaa Asp Xaa Xaa Xaa His Xaa Tyr Xaa
Tyr Xaa Xaa Xaa1 5 10 1548315PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 483Asp Xaa Xaa Pro His Xaa
Asp Gly Pro His Xaa Tyr Xaa Glu Xaa1 5 10 1548495PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
484Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr1
5 10 15Ser Leu Leu Ile Ser Trp Gln Val Pro Arg Pro Met Tyr Gln Arg
Tyr 20 25 30Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val
Gln Glu 35 40 45Phe Thr Val Pro Gly Gly Val Arg Thr Ala Thr Ile Ser
Gly Leu Lys 50 55 60Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val
Thr Asp Tyr Met65 70 75 80His Ser Glu Tyr Arg Gln Tyr Pro Ile Ser
Ile Asn Tyr Arg Thr 85 90 9548594PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 485Val Ser Asp Val Pro
Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr1 5 10 15Ser Leu Leu Ile
Ser Trp Gln Val Pro Arg Pro Met Tyr Gln Tyr Tyr 20 25 30Arg Ile Thr
Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40 45Thr Val
Pro Gly Gly Val Arg Thr Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60Gly
Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Asp Tyr Met His65 70 75
80Ser Glu Tyr Arg Gln Tyr Pro Ile Ser Ile Asn Tyr Arg Thr 85
90486102PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 486Val Ser Asp Val Pro Arg Asp Leu Glu Val
Val Ala Ala Thr Pro Thr1 5 10 15Ser Leu Leu Ile Ser Trp Gln Val Pro
Arg Pro Met Tyr Gln Arg Tyr 20 25 30Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln Glu 35 40 45Phe Thr Val Pro Gly Gly Val
Arg Thr Ala Thr Ile Ser Gly Leu Lys 50 55 60Pro Gly Val Asp Tyr Thr
Ile Thr Val Tyr Ala Val Thr Asp Tyr Met65 70 75 80His Ser Glu Tyr
Arg Gln Tyr Pro Ile Ser Ile Asn Tyr Arg Thr Glu 85 90 95Ile Asp Lys
Pro Cys Gln 1004876PRTArtificial SequenceDescription of Artificial
Sequence Synthetic 6xHis tag 487His His His His His His1
548818PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Peptide 488Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro
Ala Pro Ala Pro Ala1 5 10 15Pro Ala4894PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 489Gly
Ser Gly Cys1490109PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 490Met Gly Val Ser Asp Val Pro Arg
Asp Leu Glu Val Val Ala Ala Thr1 5 10 15Pro Thr Ser Leu Leu Ile Ser
Trp Gln Val Pro Arg Pro Met Tyr Gln 20 25 30Tyr Tyr Arg Ile Thr Tyr
Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45Glu Phe Thr Val Pro
Gly Gly Val Arg Thr Ala Thr Ile Ser Gly Leu 50 55 60Lys Pro Gly Val
Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Asp Tyr65 70 75 80Met His
Ser Glu Tyr Arg Gln Tyr Pro Ile Ser Ile Asn Tyr Arg Thr 85 90 95Glu
Ile Asp Lys Pro Ser Gln His His His His His His 100
10549119PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 491Phe Lys Gly Asp Ser Phe Thr Arg Thr Pro Pro
Leu Asp Pro Arg Glu1 5 10 15Leu Glu Ile492100PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
492Glu Glu Lys Lys Val Cys Gln Gly Thr Ser Asn Lys Leu Thr Gln Leu1
5 10 15Gly Thr Phe Glu Asp His Phe Leu Ser Leu Gln Arg Met Phe Asn
Asn 20 25 30Cys Glu Val Val Leu Gly Asn Leu Glu Ile Thr Tyr Val Gln
Arg Asn 35 40 45Tyr Asp Leu Ser Phe Leu Lys Thr Ile Gln Glu Val Ala
Gly Tyr Val 50 55 60Leu Ile Ala Leu Asn Thr Val Glu Arg Ile Pro Leu
Glu Asn Leu Gln65 70 75 80Ile Ile Arg Gly Asn Met Tyr Tyr Glu Asn
Ser Tyr Ala Leu Ala Val 85 90 95Leu Ser Asn Tyr
10049312PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 493Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His
Cys1 5 1049416PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 494Gly Ser Gly Ser Gly Ser Gly Ser Gly
Ser Gly Ser Gly Ser Gly Ser1 5 10 15495342DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
495atgggagttt ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc
caccagcctg 60ctgatcagct ggaaaacaga accaggccgc caccaatatt accgcatcac
ttacggcgaa 120acaggaggca atagccctgt ccaggagttc actgtgcctg
gtggtgttcg tacagctacc 180atcagcggcc ttaaacctgg cgttgattat
accatcactg tgtatgctgt cactgactgg 240tacaacctgg tttctcatga
atacgtatac catactaccc caatttccat taattaccgc 300acagaaattg
acaaaccatc ccagcaccat caccaccacc ac 342
* * * * *